Synthetic brassica-derived chloroplast transit peptides

ABSTRACT

This disclosure concerns compositions and methods for targeting peptides, polypeptides, and proteins to plastids of plastid-containing cells. In some embodiments, the disclosure concerns chloroplast transit peptides that may direct a polypeptide to a plastid, and nucleic acid molecules encoding the same. In some embodiments, the disclosure concerns methods for producing a transgenic plant material (e.g., a transgenic plant) comprising a chloroplast transit peptide, as well as plant materials produced by such methods, and plant commodity products produced therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/352,229 filed Nov. 15, 2016, which is a continuation of U.S. patentapplication Ser. No. 13/757,613 filed Feb. 1, 2013, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 61/593,555 filedFeb. 1, 2012, and also to U.S. Provisional Patent Application Ser. No.61/625,222, filed Apr. 17, 2012, the disclosures of each of which ishereby incorporated herein in its entirety by this reference.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS ASCII TEXT FILE

Pursuant to 37 C.F.R. § 1.821(c) or (e), a file containing an ASCII textversion of the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

This disclosure relates to compositions and methods for geneticallyencoding and expressing polypeptides that are targeted to plastids ofplastid-containing cells. In certain embodiments, the disclosure relatesto amino acid sequences that target polypeptides to chloroplasts (e.g.,of higher plants), and/or nucleic acid molecules encoding the same. Incertain embodiments, the disclosure relates to chimeric polypeptidescomprising an amino acid sequence that controls the transit of thechimeric polypeptides to plastids, and/or nucleic acid moleculesencoding the same.

BACKGROUND

Plant cells contain distinct subcellular organelles, referred togenerally as “plastids,” that are delimited by characteristic membranesystems and perform specialized functions within the cell. Particularplastids are responsible for photosynthesis, as well as the synthesisand storage of certain chemical compounds. All plastids are derived fromproplastids that are present in the meristematic regions of the plant.Proplastids may develop into, for example: chloroplasts, etioplasts,chromoplasts, gerontoplasts, leucoplasts, amyloplasts, elaioplasts, andproteinoplasts. Plastids exist in a semi-autonomous fashion within thecell, containing their own genetic system and protein synthesismachinery, but relying upon a close cooperation with thenucleo-cytoplasmic system in their development and biosyntheticactivities.

In photosynthetic leaf cells of higher plants, the most conspicuousplastids are the chloroplasts. The most essential function ofchloroplasts is the performance of the light-driven reactions ofphotosynthesis. But, chloroplasts also carry out many other biosyntheticprocesses of importance to the plant cell. For example, all of thecell's fatty acids are made by enzymes located in the chloroplaststroma, using the ATP, NAOPH, and carbohydrates readily available there.Moreover, the reducing power of light-activated electrons drives thereduction of nitrite (NO₂ ⁻) to ammonia (NH₃) in the chloroplast; thisammonia provides the plant with nitrogen required for the synthesis ofamino acids and nucleotides.

The chloroplast also takes part in processes of particular importance inthe agrochemical industry. For example, it is known that many herbicidesact by blocking functions which are performed within the chloroplast.Recent studies have identified the specific target of severalherbicides. For instance, triazine-derived herbicides inhibitphotosynthesis by displacing a plastoquinone molecule from its bindingsite in the 32 kD polypeptide of the photosystem II. This 32 kDpolypeptide is encoded in the chloroplast genome and synthesized by theorganelle machinery. Mutant plants have been obtained which areresistant to triazine herbicides. These plants contain a mutant 32 kDpolypeptide from which the plastoquinone can no longer be displaced bytriazine herbicides. Sulfonylureas inhibit acetolactate synthase in thechloroplast. Acetolactate synthase is involved in isoleucine and valinesynthesis. Glyphosate inhibits the function of 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS), which is an enzyme involvedin the synthesis of aromatic amino acids. All these enzymes are encodedby the nuclear genome, but they are translocated into the chloroplastwhere the actual amino acid synthesis takes place.

Most chloroplast proteins are encoded in the nucleus of the plant cell,synthesized as larger precursor proteins in the cytosol, andpost-translationally imported into the chloroplast. Import across theouter and inner envelope membranes into the stroma is the major meansfor entry of proteins destined for the stroma, the thylakoid membrane,and the thylakoid lumen. Localization of imported precursor proteins tothe thylakoid membrane and thylakoid lumen is accomplished by fourdistinct mechanisms, including two that are homologous to bacterialprotein transport systems. Thus, mechanisms for protein localization inthe chloroplast are, in part, derived from the prokaryotic endosymbiont.Cline and Henry (1996), Annu. Rev. Cell. Dev. Biol. 12:1-26.

Precursor proteins destined for chloroplastic expression containN-terminal extensions known as chloroplast transit peptides (CTPs). Thetransit peptide is instrumental for specific recognition of thechloroplast surface and in mediating the post-translationaltranslocation of pre-proteins across the chloroplastic envelope and,thence, to the various sub-compartments within the chloroplast (e.g.,stroma, thylakoid, and thylakoid membrane). These N-terminal transitpeptide sequences contain all the information necessary for the importof the chloroplast protein into plastids; the transit peptide sequencesare necessary and sufficient for plastid import.

Plant genes reported to have naturally-encoded transit peptide sequencesat their N-terminus include the chloroplast small subunit ofribulose-1,5-bisphosphate caroxylase (RuBisCo) (de Castro Silva-Filho etal. (1996), Plant Mol. Biol. 30:769-80; Schnell et al. (1991), J. Biol.Chem. 266:3335-42); EPSPS (see, e.g., Archer et al. (1990), J. Bioenerg.and Biomemb. 22:789-810, and U.S. Pat. Nos. 6,867,293, 7,045,684, andRe. 36,449); tryptophan synthase (Zhao et al. (1995), J. Biol. Chem.270:6081-7); plastocyanin (Lawrence et al. (1997), J. Biol. Chem.272:20357-63); chorismate synthase (Schmidt et al. (1993), J. Biol.Chem. 268:27447-57); the light harvesting chlorophyll a/b bindingprotein (LHBP) (Lamppa et al. (1988), J. Biol. Chem. 263:14996-14999);and chloroplast protein of Arabidopsis thaliana (Lee et al. (2008),Plant Cell 20:1603-22). United States Patent Publication No. US2010/0071090 provides certain chloroplast targeting peptides fromChlamydomonas sp.

However, the structural requirements for the information encoded bychloroplast targeting peptides remains elusive, due to their high levelof sequence diversity and lack of common or consensus sequence motifs,though it is possible that there are distinct subgroups of chloroplasttargeting peptides with independent structural motifs. Lee et al.(2008), supra. Further, not all of these sequences have been useful inthe heterologous expression of chloroplast-targeted proteins in higherplants.

BRIEF SUMMARY OF THE DISCLOSURE

Described herein are compositions and methods for plastid targeting ofpolypeptides in a plant. In some embodiments, a composition comprises anucleic acid molecule comprising at least one nucleotide sequenceencoding a synthetic Brassica-derived chloroplast transit peptide (e.g.,a TraP8 peptide, and a TraP9 peptide) operably linked to a nucleotidesequence of interest. In particular embodiments, such nucleic acidmolecules may be useful for expression and targeting of a polypeptideencoded by the nucleotide sequence of interest in a monocot or dicotplant. Further described are vectors comprising a nucleic acid moleculecomprising at least one nucleotide sequence encoding a syntheticBrassica-derived chloroplast transit peptide operably linked to anucleotide sequence of interest.

In some embodiments, a nucleotide sequence encoding a syntheticBrassica-derived CTP may be a nucleotide sequence that is derived from areference nucleotide sequence obtained from a Brassica sp. gene (e.g.,B. napus, B. rapa, B. juncea, and B. carinata), or a functional variantthereof. In some embodiments, a nucleotide sequence encoding a syntheticBrassica-derived CTP may be a chimeric nucleotide sequence comprising apartial CTP-encoding nucleotide sequence from a Brassica sp. gene, or afunctional variant thereof. In specific embodiments, a nucleotidesequence encoding a synthetic Brassica-derived CTP may containcontiguous nucleotide sequences obtained from each of a referenceBrassica sp. CTP, and a CTP from a different gene of the Brassica sp., adifferent Brassica sp., or a different organism (e.g., a plant,prokaryote, and lower photosynthetic eukaryote), or or functionalvariants of any of the foregoing. In particular embodiments, acontiguous nucleotide sequence may be obtained from an orthologousnucleotide sequence of the reference Brassica CTP that is obtained froma different organism's ortholog of the reference Brassica sp. gene(e.g., a different Brassica sp. genome). In these and furtherembodiments, a nucleotide sequence encoding a synthetic Brassica-derivedCTP may be a chimeric nucleotide sequence comprising more than oneCTP-encoding nucleotide sequence.

In some examples, a nucleotide sequence encoding a syntheticBrassica-derived CTP may be a chimeric nucleotide sequence comprising apartial CTP nucleotide sequence from either of B. napus and B. rapa, orfunctional variants thereof. In specific examples, a nucleotide sequenceencoding a synthetic Brassica-derived CTP may contain contiguousnucleotide sequences obtained from each of B. napus and B. rapa, orfunctional variants thereof.

In some embodiments, a composition comprises a nucleic acid moleculecomprising at least one Brassica-derived means for targeting apolypeptide to a chloroplast. Further described are nucleic acidmolecules comprising a nucleic acid molecule comprising at least oneBrassica-derived means for targeting a polypeptide to a chloroplastoperably linked to a nucleotide sequence of interest. In particularembodiments, such nucleic acid molecules may be useful for expressionand targeting of a polypeptide encoded by the nucleotide sequence ofinterest in a monocot or dicot plant. For the purposes of the presentdisclosure, a Brassica-derived means for targeting a polypeptide to achloroplast refers to particular synthetic nucleotide sequences. Inparticular embodiments, a Brassica-derived means for targeting apolypeptide to a chloroplast is selected from the group consisting ofthe nucleotide sequences encoding the polypeptides referred to herein asTraP8 and TraP9.

Also described herein are plant materials (for example and withoutlimitation, plants, plant tissues, and plant cells) comprising a nucleicacid molecule comprising at least one nucleotide sequence encoding asynthetic Brassica-derived CTP operably linked to a nucleotide sequenceof interest. In some embodiments, a plant material may have such anucleic acid molecule stably integrated in its genome. In someembodiments, a plant material may transiently express a product of anucleic acid molecule comprising at least one nucleotide sequenceencoding a synthetic Brassica-derived CTP operably linked to anucleotide sequence of interest. In some embodiments, the plant materialis a plant cell from which a plant cannot be regenerated.

Methods are also described for expressing a nucleotide sequence in aplastid-containing cell (e.g., a plant) in a plastid (e.g., achloroplast) of the plastid-containing cell. In particular embodiments,a nucleic acid molecule comprising at least one nucleotide sequenceencoding a synthetic Brassica-derived CTP operably linked to anucleotide sequence of interest may be used to transform a plant cell,such that a precursor fusion polypeptide comprising the syntheticBrassica-derived CTP fused to an expression product of the nucleotidesequence of interest is produced in the cytoplasm of the plant cell, andthe fusion polypeptide is then transported in vivo into a chloroplast ofthe plant cell. In some embodiments, the plant cell is not capable ofregeneration to a plant.

Further described are methods for the production of a transgenic plantcomprising a nucleic acid molecule comprising at least one nucleotidesequence encoding a synthetic Brassica-derived CTP operably linked to anucleotide sequence of interest. Also described are plant commodityproducts (e.g., seeds) produced from such transgenic plants. In someembodiments, these transgenic plants or plant commodity products containtransgenic cells from which a plant cannot be regenerated.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an mRNA molecule that is representative of particularexamples of synthetic Brassica-derived CTP-encoding nucleotide sequences(for example, for TraP8 and TraP9) operably linked to a nucleotidesequence of interest. In some embodiments, an mRNA molecule (such as theone shown) may be transcribed from a DNA molecule comprising an openreading frame including the synthetic Brassica-derived CTP-encodingsequence operably linked to the nucleotide sequence of interest. Thenucleotide sequence of interest may be, in some embodiments, a sequenceencoding a peptide of interest, for example and without limitation, amarker gene product or peptide to be targeted to a plastid.

FIG. 2 illustrates a plasmid map of pDAB101977.

FIG. 3 illustrates a plasmid map of pDAB101978.

FIG. 4 illustrates a plasmid map of pDAB101908.

FIG. 5 includes a microscopy image showing that TraP8-YFP infiltratedinto tobacco leaf tissue was translocated into the chloroplasts of thetobacco leaf tissue.

FIG. 6 includes a microscopy image showing that TraP9-YFP infiltratedinto tobacco leaf tissue was translocated into the chloroplasts of thetobacco leaf tissue.

FIG. 7 includes a microscopy image showing that non-targeted YFPcontrols that were infiltrated into tobacco leaf tissue were notincorporated into the chloroplasts of the tobacco leaf tissue.

FIG. 8 illustrates a plasmid map of pDAB106597.

FIG. 9 includes a microscopy image of the TraP8-YFP constructtransformed into maize protoplasts showing the translocation into thechloroplasts of the maize protoplast.

FIG. 10 illustrates a plasmid map of pDAB105526.

FIG. 11 illustrates a plasmid map of pDAB105527.

FIG. 12 illustrates a plasmid map of pDAB109807.

FIG. 13 illustrates a plasmid map of pDAB107687.

FIG. 14 illustrates a plasmid map of pDAB111481.

FIG. 15 illustrates a plasmid map of pDAB111479.

FIG. 16 illustrates a plasmid map of pDAB111338.

FIG. 17 illustrates a plasmid map of pDAB112710.

FIG. 18 includes an alignment of the predicted chloroplast transitpeptides for the EPSPS protein from Brassica napus (SEQ ID NO:1) andBrassica rapa (SEQ ID NO:2). The asterisk indicates where the sequenceswere split and recombined to form TraP8 and Trap9.

FIG. 19 illustrates a plasmid map of pDAB107527.

FIG. 20 illustrates a plasmid map of pDAB105530.

FIG. 21 illustrates a plasmid map of pDAB105531.

FIG. 22 illustrates a plasmid map of pDAB105532.

FIG. 23 illustrates a plasmid map of pDAB105533.

FIG. 24 illustrates a plasmid map of pDAB105534.

FIG. 25 illustrates a plasmid map of pDAB107532.

FIG. 26 illustrates a plasmid map of pDAB107534.

FIG. 27 illustrates a plasmid map of pDAB107533.

FIG. 28 illustrates a plasmid map of pDAB4104.

FIG. 29 illustrates a plasmid map of pDAB102715.

FIG. 30 illustrates a plasmid map of pDAB102716.

FIG. 31 illustrates a plasmid map of pDAB102717.

FIG. 32 illustrates a plasmid map of pDAB102785.

FIG. 33 illustrates a plasmid map of pDAB102719.

FIG. 34 illustrates a plasmid map of pDAB102718.

FIG. 35 illustrates a plasmid map of pDAB107663.

FIG. 36 illustrates a plasmid map of pDAB107664.

FIG. 37 illustrates a plasmid map of pDAB107665.

FIG. 38 illustrates a plasmid map of pDAB107666.

FIG. 39 illustrates a plasmid map of pDAB109812.

FIG. 40 illustrates a plasmid map of pDAB101556.

FIG. 41 illustrates a plasmid map of pDAB107698.

FIG. 42 illustrates a plasmid map of pDAB108384.

FIG. 43 illustrates a plasmid map of pDAB108385.

FIG. 44 illustrates a plasmid map of pDAB108386.

FIG. 45 illustrates a plasmid map of pDAB108387.

SEQUENCE LISTING

The nucleic acid sequences listed in the accompanying sequence listingare shown using standard letter abbreviations for nucleotide bases, asdefined in 37 C.F.R. § 1.822. Only one strand of each nucleic acidsequence is shown, but the complementary strand is understood to beincluded by any reference to the displayed strand. In the accompanyingsequence listing:

SEQ ID NO:1 shows the amino acid of a Brassica napus EPSPS chloroplasttransit peptide.

SEQ ID NO:2 shows the amino acid of a Brassica rapa EPSPS chloroplasttransit peptide.

SEQ ID NO:3 shows the amino acid of a TraP8 chimeric chloroplast transitpeptide.

SEQ ID NO:4 shows the amino acid of a TraP9 chimeric chloroplast transitpeptide.

SEQ ID NO:5 shows a polynucleotide sequence encoding a TraP8 chimericchloroplast transit peptide.

SEQ ID NO:6 shows a polynucleotide sequence encoding a TraP9 chimericchloroplast transit peptide.

SEQ ID NO:7 shows a polynucleotide sequence encoding a linker sequence.

SEQ ID NO:8 shows a polynucleotide sequence encoding a TraP8 v2 chimericchloroplast transit peptide.

SEQ ID NO:9 shows a polynucleotide sequence encoding a TraP9 v2 chimericchloroplast transit peptide.

SEQ ID NO:10 shows a polynucleotide sequence encoding a cry2aa gene.

SEQ ID NO:11 shows a polynucleotide sequence encoding a wp3ab1v6 gene.

SEQ ID NO:12 shows a polynucleotide sequence encoding a wp3ab1v7 gene.

SEQ ID NO:13 shows a peptide having the amino acid sequence,Ser-Val-Ser-Leu.

SEQ ID NO:14 shows a polynucleotide sequence encoding the Brassica napusEPSPS chloroplast transit peptide of SEQ ID NO:1.

SEQ ID NO:15 shows a polynucleotide sequence encoding the Brassica rapaEPSPS chloroplast transit peptide of SEQ ID NO:2.

DETAILED DESCRIPTION I. Overview of Several Embodiments

A chloroplast transit peptide (CTP) (or plastid transit peptide)functions co-translationally or post-translationally to direct apolypeptide comprising the CTP to a plastid (e.g., a chloroplast). Insome embodiments of the invention, either endogenous chloroplastproteins or heterologous proteins may be directed to a chloroplast byexpression of such a protein as a larger precursor polypeptidecomprising a CTP. In particular embodiments, the CTP may be derived froma nucleotide sequence obtained from a Brassica sp. gene, for example andwithout limitation, by incorporating at least one contiguous sequencefrom a orthologous gene obtained from a different organism, or afunctional variant thereof.

In an exemplary embodiment, nucleic acid sequences, each encoding a CTP,were isolated from EPSPS gene sequences obtained from Brassica napus(NCBI Database Accession No. P17688) and Brassica rapa (NCBI DatabaseAccession No. AAS80163). The CTP-encoding nucleic acid sequences wereisolated by analyzing the EPSPS gene sequence with the ChloroPprediction server. Emanuelsson et al. (1999), Protein Science 8:978-84(available at cbs.dtu.dk/services/ChloroP). The predicted proteinproducts of the isolated CTP-encoding sequences are approximately 60-70amino acid-long transit peptides. In this example, the native B. napusCTP was used as a reference sequence to design exemplary syntheticBrassica-derived CTPs by fusing contiguous sequences from the other CTPsat a particular position in the B. napus CTP. This design processillustrates the development of a novel synthetic CTP, according to someaspects, from a Brassica sp. nucleic acid sequence. These exemplarysynthetic Brassica-derived CTPs are referred to throughout thisdisclosure as TraP8 and TraP9. These exemplary synthetic TraPs weretested for plastid-targeting function and were found to exhibit plastidtargeting that was at least as favorable as that observed for the nativeBrassica sequences individually.

In a further exemplary embodiment, nucleic acid sequences, each encodinga synthetic TraP peptide of the invention, were synthesizedindependently and operably linked to a nucleic acid sequence encoding ayellow fluorescent protein (YFP) to produce synthetic nucleic acidmolecules, each encoding a chimeric TraP:YFP fusion polypeptide. Suchnucleic acid molecules, each encoding a chimeric TraP:YFP polypeptide,were each introduced into a binary vector, such that eachTraP:YFP-encoding nucleic acid sequence was operably linked to an AtUbi10 promoter.

In yet a further exemplary embodiment, binary vectors comprising aTraP:YFP-encoding nucleic acid sequence operably linked to an AtUbi 10promoter each were independently, transiently transformed into tobacco(Nicotiana benthamiana) via Agrobacterium-mediated transformation.Confocal microscopy and Western blot analysis confirmed that each TraPsuccessfully targeted YFP to tobacco chloroplasts.

In a further exemplary embodiment, nucleic acid sequences, each encodinga synthetic TraP peptide of the invention, were synthesizedindependently and operably linked to a nucleic acid sequence encoding anagronomically important gene sequence. The TraP sequences were fused toherbicide tolerant traits (e.g. dgt-28 and dgt-14) to produce syntheticnucleic acid molecules, each encoding a chimeric TraP:DGT-28 orTraP:DGT-14 fusion polypeptide. Such nucleic acid molecules, eachencoding a chimeric TraP:DGT-28 or TraP:DGT-14 polypeptide, were eachintroduced into a binary vector, such that each TraP:dgt-28 orTraP:dgt-14-encoding nucleic acid sequence was operably linked to apromoter and other gene regulatory elements. The binary containing theTraP:dgt-28 or TraP:dgt-14-encoding nucleic acid sequence was used totransform varopis plant species. The transgenic plants were assayed forherbicide tolerance as a result of the expression and translocation ofthe DGT-28 or DGT-14 enzymes to the chloroplast.

In a further exemplary embodiment, nucleic acid sequences, each encodinga synthetic TraP peptide of the invention, were synthesizedindependently and operably linked to a nucleic acid sequence encoding anagronomically important gene sequence. The TraP sequences were fused togenes conferring insect tolerance traits (e.g. cry2Aa and vip3ab1) toproduce synthetic nucleic acid molecules, each encoding a chimericTraP:Cry2Aa or TraP:Vip3Ab1 fusion polypeptide. Such nucleic acidmolecules, each encoding a chimeric TraP:Cry2Aa or TraP: Vip3Ab1polypeptide, were each introduced into a binary vector, such that eachTraP: Cry2Aa or TraP: Vip3Ab1—encoding nucleic acid sequence wasoperably linked to a promoter and other gene regulatory elements. Thebinary containing the TraP: Cry2Aa or TraP: Vip3Ab1-encoding nucleicacid-sequence was used to transform various plant species. Thetransgenic plants were bioassayed for insect resistance as a result ofthe expression and translocation of the Cry2Aa or Vip3Ab1 enzymes to thechloroplast.

In view of the aforementioned detailed working examples, syntheticBrassica-derived CTP sequences of the invention, and nucleic acidsencoding the same, may be used to direct any polypeptide to a plastid ina broad range of plastid-containing cells. For example, by methods madeavailable to those of skill in the art by the present disclosure, achimeric polypeptide comprising a synthetic Brassica-derived CTPsequence fused to the N-terminus of any second peptide sequence may beintroduced into (or expressed in) a plastid-containing host cell forplastid targeting of the second peptide sequence. Thus, in particularembodiments, a TraP peptide of the invention may provide increasedefficiency of import and processing of a peptide for which plastidexpression is desired, when compared to a native CTP.

II. Abbreviations

-   -   CTP chloroplast transit peptide    -   Bt Bacillus thuringiensis    -   EPSPS 3-enolpyruvylshikimate-5-phosphate synthetase    -   YFP yellow fluorescent protein    -   T_(i) tumor-inducing (plasmids derived from A. tumefaciens)    -   T-DNA transfer DNA

III. Terms

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Chloroplast transit peptide: As used herein, the term “chloroplasttransit peptide” (CTP) (or “plastid transit peptide”) may refer to anamino acid sequence that, when present at the N-terminus of apolypeptide, directs the import of the polypeptide into a plastid of aplastid-containing cell (e.g., a plant cell, such as in a whole plant orplant cell culture). A CTP is generally necessary and sufficient todirect the import of a protein into a plastid (e.g., a primary,secondary, or tertiary plastid, such as a chloroplast) of a host cell. Aputative chloroplast transit peptide may be identified by one of severalavailable algorithms (e.g., PSORT, and ChloroP (available atcbs.dtu.dk/services/ChloroP)). ChloroP may provide particularly goodprediction of CTPs. Emanuelsson et al. (1999), Protein Science 8:978-84.However, prediction of functional CTPs is not achieved at 100%efficiency by any existing algorithm. Therefore, it is important toverify that an identified putative CTP does indeed function as intendedin, e.g., an in vitro, or in vivo methodology.

Chloroplast transit peptides may be located at the N-terminus of apolypeptide that is imported into a plastid. The CTP may facilitate co-or post-translational transport of a polypeptide comprising the CTP intothe plastid. Chloroplast transit peptides typically comprise betweenabout 40 and about 100 amino acids, and such CTPs have been observed tocontain certain common characteristics. For example: CTPs contain veryfew, if any, negatively charged amino acids (such as aspartic acid,glutamic acid, asparagines, or glutamine); the N-terminal regions ofCTPs lack charged amino acids, glycine, and proline; the central regionof a CTP also is likely to contain a very high proportion of basic orhydroxylated amino acids (such as serine and threonine); and theC-terminal region of a CTP is likely to be rich in arginine, and havethe ability to comprise an amphipathic beta-sheet structure. Plastidproteases may cleave the CTP from the remainder of a polypeptidecomprising the CTP after importation of the polypeptide into theplastid.

Contact: As used herein, the term “contact with” or “uptake by” a cell,tissue, or organism (e.g., a plant cell; plant tissue; and plant), withregard to a nucleic acid molecule, includes internalization of thenucleic acid molecule into the organism, for example and withoutlimitation: contacting the organism with a composition comprising thenucleic acid molecule; and soaking of organisms with a solutioncomprising the nucleic acid molecule.

Endogenous: As used herein, the term “endogenous” refers to substances(e.g., nucleic acid molecules and polypeptides) that originate fromwithin a particular organism, tissue, or cell. For example, an“endogenous” polypeptide expressed in a plant cell may refer to apolypeptide that is normally expressed in cells of the same type fromnon-genetically engineered plants of the same species. In some examples,an endogenous gene (e.g., an EPSPS gene) from a Brassica sp. may be usedto obtain a reference Brassica CTP sequence.

Expression: As used herein, “expression” of a coding sequence (forexample, a gene or a transgene) refers to the process by which the codedinformation of a nucleic acid transcriptional unit (including, e.g.,genomic DNA or cDNA) is converted into an operational, non-operational,or structural part of a cell, often including the synthesis of aprotein. Gene expression can be influenced by external signals; forexample, exposure of a cell, tissue, or organism to an agent thatincreases or decreases gene expression. Expression of a gene can also beregulated anywhere in the pathway from DNA to RNA to protein. Regulationof gene expression occurs, for example, through controls acting ontranscription, translation, RNA transport and processing, degradation ofintermediary molecules such as mRNA, or through activation,inactivation, compartmentalization, or degradation of specific proteinmolecules after they have been made, or by combinations thereof. Geneexpression can be measured at the RNA level or the protein level by anymethod known in the art, for example and without limitation: Northernblot; RT-PCR; Western blot; or in vitro; in situ; and in vivo proteinactivity assay(s).

Genetic material: As used herein, the term “genetic material” includesall genes, and nucleic acid molecules, such as DNA and RNA.

Heterologous: As used herein, the term “heterologous” refers tosubstances (e.g., nucleic acid molecules and polypeptides) that do notoriginate from within a particular organism, tissue, or cell. Forexample, a “heterologous” polypeptide expressed in a plant cell mayrefer to a polypeptide that is not normally expressed in cells of thesame type from non-genetically engineered plants of the same species(e.g., a polypeptide that is expressed in different cells of the sameorganism or cells of a different organism).

Isolated: As used herein, the term “isolated” refers to molecules (e.g.,nucleic acid molecules and polypeptides) that are substantiallyseparated or purified away from other molecules of the same type (e.g.,other nucleic acid molecules and other polypeptides) with which themolecule is normally associated in the cell of the organism in which themolecule naturally occurs. For example, an isolated nucleic acidmolecule may be substantially separated or purified away fromchromosomal DNA or extrachromosomal DNA in the cell of the organism inwhich the nucleic acid molecule naturally occurs. Thus, the termincludes recombinant nucleic acid molecules and polypeptides that arebiochemically purified such that other nucleic acid molecules,polypeptides, and cellular components are removed. The term alsoincludes recombinant nucleic acid molecules, chemically-synthesizednucleic acid molecules, and recombinantly produced polypeptides.

The term “substantially purified,” as used herein, refers to a moleculethat is separated from other molecules normally associated with it inits native state. A substantially purified molecule may be thepredominant species present in a composition. A substantially purifiedmolecule may be, for example, at least 60% free, at least 75% free, orat least 90% free from other molecules besides a solvent present in anatural mixture. The term “substantially purified” does not refer tomolecules present in their native state.

Nucleic acid molecule: As used herein, the term “nucleic acid molecule”refers to a polymeric form of nucleotides, which may include both senseand anti-sense strands of RNA, cDNA, genomic DNA, and synthetic formsand mixed polymers of the above. A nucleotide may refer to aribonucleotide, deoxyribonucleotide, or a modified form of either typeof nucleotide. A “nucleic acid molecule” as used herein is synonymouswith “nucleic acid” and “polynucleotide.” A nucleic acid molecule isusually at least 10 bases in length, unless otherwise specified. Theterm includes single- and double-stranded forms of DNA. Nucleic acidmolecules include dimeric (so-called in tandem) forms, and thetranscription products of nucleic acid molecules. A nucleic acidmolecule can include either or both naturally occurring and modifiednucleotides linked together by naturally occurring and/or non-naturallyoccurring nucleotide linkages.

Nucleic acid molecules may be modified chemically or biochemically, ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of skill in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications (e.g., uncharged linkages: for example, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.;charged linkages: for example, phosphorothioates, phosphorodithioates,etc.; pendent moieties: for example, peptides; intercalators: forexample, acridine, psoralen, etc.; chelators; alkylators; and modifiedlinkages: for example, alpha anomeric nucleic acids, etc.). The term“nucleic acid molecule” also includes any topological conformation,including single-stranded, double-stranded, partially duplexed,triplexed, hairpinned, circular, and padlocked conformations.

As used herein with respect to DNA, the term “coding sequence,”“structural nucleotide sequence,” or “structural nucleic acid molecule”refers to a nucleotide sequence that is ultimately translated into apolypeptide, via transcription and mRNA, when placed under the controlof appropriate regulatory sequences. With respect to RNA, the term“coding sequence” refers to a nucleotide sequence that is translatedinto a peptide, polypeptide, or protein. The boundaries of a codingsequence are determined by a translation start codon at the 5′-terminusand a translation stop codon at the 3′-terminus. Coding sequencesinclude, but are not limited to: genomic DNA; cDNA; ESTs; andrecombinant nucleotide sequences.

In some embodiments, the invention includes nucleotide sequences thatmay be isolated, purified, or partially purified, for example, usingseparation methods such as, for example, ion-exchange chromatography; byexclusion based on molecular size or by affinity; by fractionationtechniques based on solubility in different solvents; and methods ofgenetic engineering such as amplification, cloning, and subcloning.

Sequence identity: The term “sequence identity” or “identity,” as usedherein in the context of two nucleic acid or polypeptide sequences, mayrefer to the residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window.

As used herein, the term “percentage of sequence identity” may refer tothe value determined by comparing two optimally aligned sequences (e.g.,nucleic acid sequences, and amino acid sequences) over a comparisonwindow, wherein the portion of the sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleotide oramino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the comparison window, and multiplying the resultby 100 to yield the percentage of sequence identity.

Methods for aligning sequences for comparison are well-known in the art.Various programs and alignment algorithms are described in, for example:Smith and Waterman (1981), Adv. Appl. Math. 2:482; Needleman and Wunsch(1970), J. Mol. Biol. 48:443; Pearson and Lipman (1988), Proc. Natl.Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988), Gene 73:237-44;Higgins and Sharp (1989), CABIOS 5:151-3; Corpet et al. (1988), NucleicAcids Res. 16:10881-90; Huang et al. (1992), Comp. Appl. Biosci.8:155-65; Pearson et al. (1994), Methods Mol. Biol. 24:307-31; Tatianaet al. (1999), FEMS Microbiol. Lett. 174:247-50. A detailedconsideration of sequence alignment methods and homology calculationscan be found in, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10.

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™; Altschul et al. (1990)) is available fromseveral sources, including the National Center for BiotechnologyInformation (Bethesda, Md.), and on the internet, for use in connectionwith several sequence analysis programs. A description of how todetermine sequence identity using this program is available on theinternet under the “help” section for BLAST™. For comparisons of nucleicacid sequences, the “Blast 2 sequences” function of the BLAST™ (Blastn)program may be employed using the default BLOSUM62 matrix set to defaultparameters. Nucleic acid sequences with even greater similarity to thereference sequences will show increasing percentage identity whenassessed by this method.

Specifically hybridizable/Specifically complementary: As used herein,the terms “Specifically hybridizable” and “specifically complementary”are terms that indicate a sufficient degree of complementarity, suchthat stable and specific binding occurs between the nucleic acidmolecule and a target nucleic acid molecule. Hybridization between twonucleic acid molecules involves the formation of an anti-parallelalignment between the nucleic acid sequences of the two nucleic acidmolecules. The two molecules are then able to form hydrogen bonds withcorresponding bases on the opposite strand to form a duplex moleculethat, if it is sufficiently stable, is detectable using methods wellknown in the art. A nucleic acid molecule need not be 100% complementaryto its target sequence to be specifically hybridizable. However, theamount of sequence complementarity that must exist for hybridization tobe specific is a function of the hybridization conditions used.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ and/or Mg⁺⁺ concentration) of thehybridization buffer will determine the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are known to those of ordinary skill in the art, and arediscussed, for example, in Sambrook et al. (ed.) Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames andHiggins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985.Further detailed instruction and guidance with regard to thehybridization of nucleic acids may be found, for example, in Tijssen,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” in Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2, Elsevier, N Y, 1993; and Ausubel et al., Eds., CurrentProtocols in Molecular Biology, Chapter 2, Greene Publishing andWiley-Interscience, N Y, 1995.

As used herein, “stringent conditions” encompass conditions under whichhybridization will only occur if there is less than 20% mismatch betweenthe hybridization molecule and a homologous sequence within the targetnucleic acid molecule. “Stringent conditions” include further particularlevels of stringency. Thus, as used herein, “moderate stringency”conditions are those under which molecules with more than 20% sequencemismatch will not hybridize; conditions of “high stringency” are thoseunder which sequences with more than 10% mismatch will not hybridize;and conditions of “very high stringency” are those under which sequenceswith more than 5% mismatch will not hybridize.

The following are representative, non-limiting hybridization conditions.

-   -   High Stringency condition (detects sequences that share at least        90% sequence identity): Hybridization in 5×SSC buffer at 65° C.        for 16 hours; wash twice in 2×SSC buffer at room temperature for        15 minutes each; and wash twice in 0.5×SSC buffer at 65° C. for        20 minutes each.    -   Moderate Stringency condition (detects sequences that share at        least 80% sequence identity): Hybridization in 5×-6×SSC buffer        at 65-70° C. for 16-20 hours; wash twice in 2×SSC buffer at room        temperature for 5-20 minutes each; and wash twice in 1×SSC        buffer at 55-70° C. for 30 minutes each.    -   Non-stringent control condition (sequences that share at least        50% sequence identity will hybridize): Hybridization in 6×SSC        buffer at room temperature to 55° C. for 16-20 hours; wash at        least twice in 2×-3×SSC buffer at room temperature to 55° C. for        20-30 minutes each.

As used herein, the term “substantially homologous” or “substantialhomology,” with regard to a contiguous nucleic acid sequence, refers tocontiguous nucleotide sequences that hybridize under stringentconditions to the reference nucleic acid sequence. For example, nucleicacid sequences that are substantially homologous to a reference nucleicacid sequence are those nucleic acid sequences that hybridize understringent conditions (e.g., the Moderate Stringency conditions setforth, supra) to the reference nucleic acid sequence. Substantiallyhomologous sequences may have at least 80% sequence identity. Forexample, substantially homologous sequences may have from about 80% to100% sequence identity, such as about 81%; about 82%; about 83%; about84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%;about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100%. Theproperty of substantial homology is closely related to specifichybridization. For example, a nucleic acid molecule is specificallyhybridizable when there is a sufficient degree of complementarity toavoid non-specific binding of the nucleic acid to non-target sequencesunder conditions where specific binding is desired, for example, understringent hybridization conditions.

As used herein, the term “ortholog” (or “orthologous”) refers to a genein two or more species that has evolved from a common ancestralnucleotide sequence, and may retain the same function in the two or morespecies.

As used herein, two nucleic acid sequence molecules are said to exhibit“complete complementarity” when every nucleotide of a sequence read inthe 5′ to 3′ direction is complementary to every nucleotide of the othersequence when read in the 3′ to 5′ direction. A nucleotide sequence thatis complementary to a reference nucleotide sequence will exhibit asequence identical to the reverse complement sequence of the referencenucleotide sequence. These terms and descriptions are well defined inthe art and are easily understood by those of ordinary skill in the art.

When determining the percentage of sequence identity between amino acidsequences, it is well-known by those of skill in the art that theidentity of the amino acid in a given position provided by an alignmentmay differ without affecting desired properties of the polypeptidescomprising the aligned sequences. In these instances, the percentsequence identity may be adjusted to account for similarity betweenconservatively substituted amino acids. These adjustments are well-knownand commonly used by those of skill in the art. See, e.g., Myers andMiller (1988), Computer Applications in Biosciences 4:11-7.

Embodiments of the invention include functional variants of exemplaryplastid transit peptide amino acid sequences, and nucleic acid sequencesencoding the same. A functional variant of an exemplary transit peptidesequence may be, for example, a fragment of an exemplary transit peptideamino acid sequence (such as an N-terminal or C-terminal fragment), or amodified sequence of a full-length exemplary transit peptide amino acidsequence or fragment of an exemplary transit peptide amino acidsequence. An exemplary transit peptide amino acid sequence may bemodified in some embodiments be introducing one or more conservativeamino acid substitutions. A “conservative” amino acid substitution isone in which the amino acid residue is replaced by an amino acid residuehaving a similar functional side chain, similar size, and/or similarhydrophobicity. Families of amino acids that may be used to replaceanother amino acid of the same family in order to introduce aconservative substitution are known in the art. For example, these aminoacid families include: Basic amino acids (e.g., lysine, arginine, andhistidine); acidic amino acids (e.g., aspartic acid and glutamic acid);uncharged (at physiological pH) polar amino acids (e.g., glycine,asparagines, glutamine, serine, threonine, tyrosine, and cytosine);non-polar amino acids (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, and tryptophan); beta-branched aminoacids (e.g., threonine, valine, and isoleucine); and aromatic aminoacids (e.g., tyrosine, phenylalanine, tryptophan, and histidine). See,e.g., Sambrook et al. (Eds.), supra; and Innis et al., PCR Protocols: AGuide to Methods and Applications, 1990, Academic Press, NY, USA.

Operably linked: A first nucleotide sequence is “operably linked” with asecond nucleotide sequence when the first nucleotide sequence is in afunctional relationship with the second nucleotide sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.When recombinantly produced, operably linked nucleotide sequences aregenerally contiguous and, where necessary to join two protein-codingregions, in the same reading frame. However, nucleotide sequences neednot be contiguous to be operably linked.

The term, “operably linked,” when used in reference to a regulatorysequence and a coding sequence, means that the regulatory sequenceaffects the expression of the linked coding sequence. “Regulatorysequences,” or “control elements,” refer to nucleotide sequences thatinfluence the timing and level/amount of transcription, RNA processingor stability, or translation of the associated coding sequence.Regulatory sequences may include promoters; translation leadersequences; introns; enhancers; stem-loop structures; repressor bindingsequences; termination sequences; polyadenylation recognition sequences;etc. Particular regulatory sequences may be located upstream and/ordownstream of a coding sequence operably linked thereto. Also,particular regulatory sequences operably linked to a coding sequence maybe located on the associated complementary strand of a double-strandednucleic acid molecule.

When used in reference to two or more amino acid sequences, the term“operably linked” means that the first amino acid sequence is in afunctional relationship with at least one of the additional amino acidsequences. For instance, a transit peptide (e.g., a CTP) is operablylinked to a second amino acid sequence within a polypeptide comprisingboth sequences if the transit peptide affects expression or traffickingof the polypeptide or second amino acid sequence.

Promoter: As used herein, the term “promoter” refers to a region of DNAthat may be upstream from the start of transcription, and that may beinvolved in recognition and binding of RNA polymerase and other proteinsto initiate transcription. A promoter may be operably linked to a codingsequence for expression in a cell, or a promoter may be operably linkedto a nucleotide sequence encoding a signal sequence which may beoperably linked to a coding sequence for expression in a cell. A “plantpromoter” may be a promoter capable of initiating transcription in plantcells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters which initiate transcription only in certain tissues arereferred to as “tissue-specific.” A “cell type-specific” promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promotermay be a promoter which may be under environmental control. Examples ofenvironmental conditions that may initiate transcription by induciblepromoters include anaerobic conditions and the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which may be active under mostenvironmental conditions.

Any inducible promoter can be used in some embodiments of the invention.See Ward et al. (1993), Plant Mol. Biol. 22:361-366. With an induciblepromoter, the rate of transcription increases in response to an inducingagent. Exemplary inducible promoters include, but are not limited to:Promoters from the ACEI system that responds to copper; Int gene frommaize that responds to benzenesulfonamide herbicide safeners; Tetrepressor from Tn10; and the inducible promoter from a steroid hormonegene, the transcriptional activity of which may be induced by aglucocorticosteroid hormone (Schena et al. (1991), Proc. Natl. Acad.Sci. USA 88:0421).

Exemplary constitutive promoters include, but are not limited to:Promoters from plant viruses, such as the 35S promoter from CaMV;promoters from rice actin genes; ubiquitin promoters; pEMU; MAS; maizeH3 histone promoter; and the ALS promoter, Xba1/NcoI fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xba1/NcoI fragment) (International PCT Publication No. WO96/30530).

Additionally, any tissue-specific or tissue-preferred promoter may beutilized in some embodiments of the invention. Plants transformed with anucleic acid molecule comprising a coding sequence operably linked to atissue-specific promoter may produce the product of the coding sequenceexclusively, or preferentially, in a specific tissue. Exemplarytissue-specific or tissue-preferred promoters include, but are notlimited to: A root-preferred promoter, such as that from the phaseolingene; a leaf-specific and light-induced promoter such as that from cabor rubisco; an anther-specific promoter such as that from LAT52; apollen-specific promoter such as that from Zm13; and amicrospore-preferred promoter such as that from apg.

Transformation: As used herein, the term “transformation” or“transduction” refers to the transfer of one or more nucleic acidmolecule(s) into a cell. A cell is “transformed” by a nucleic acidmolecule transduced into the cell when the nucleic acid molecule becomesstably replicated by the cell, either by incorporation of the nucleicacid molecule into the cellular genome, or by episomal replication. Asused herein, the term “transformation” encompasses all techniques bywhich a nucleic acid molecule can be introduced into such a cell.Examples include, but are not limited to: transfection with viralvectors; transformation with plasmid vectors; electroporation (Fromm etal. (1986), Nature 319:791-3); lipofection (Feigner et al. (1987), Proc.Natl. Acad. Sci. USA 84:7413-7); microinjection (Mueller et al. (1978),Cell 15:579-85); Agrobacterium-mediated transfer (Fraley et al. (1983),Proc. Natl. Acad. Sci. USA 80:4803-7); direct DNA uptake; andmicroprojectile bombardment (Klein et al. (1987), Nature 327:70).

Transgene: An exogenous nucleic acid sequence. In some examples, atransgene may be a sequence that encodes a polypeptide comprising atleast one synthetic Brassica-derived CTP. In particular examples, atransgene may encode a polypeptide comprising at least one syntheticBrassica-derived CTP and at least an additional peptide sequence (e.g.,a peptide sequence that confers herbicide-resistance), for which plastidexpression is desirable. In these and other examples, a transgene maycontain regulatory sequences operably linked to a coding sequence of thetransgene (e.g., a promoter). For the purposes of this disclosure, theterm “transgenic” when used to refer to an organism (e.g., a plant),refers to an organism that comprises the exogenous nucleic acidsequence. In some examples, the organism comprising the exogenousnucleic acid sequence may be an organism into which the nucleic acidsequence was introduced via molecular transformation techniques. Inother examples, the organism comprising the exogenous nucleic acidsequence may be an organism into which the nucleic acid sequence wasintroduced by, for example, introgression or cross-pollination in aplant.

Transport: As used herein, the terms “transport(s),” “target(s),” and“transfer(s)” refers to the property of certain amino acid sequences ofthe invention that facilitates the movement of a polypeptide comprisingthe amino acid sequence from the nucleus of a host cell into a plastidof the host cell. In particular embodiments, such an amino acid sequence(i.e., a synthetic Brassica-derived CTP sequence) may be capable oftransporting about 100%, at least about 95%, at least about 90%, atleast about 85%, at least about 80%, at least about 70%, at least about60%, and/or at least about 50% of a polypeptide comprising the aminoacid sequence into plastids of a host cell.

Vector: A nucleic acid molecule as introduced into a cell, for example,to produce a transformed cell. A vector may include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. Examples of vectors include, but are not limitedto: a plasmid; cosmid; bacteriophage; or virus that carries exogenousDNA into a cell. A vector may also include one or more genes, antisensemolecules, and/or selectable marker genes and other genetic elementsknown in the art. A vector may transduce, transform, or infect a cell,thereby causing the cell to express the nucleic acid molecules and/orproteins encoded by the vector. A vector optionally includes materialsto aid in achieving entry of the nucleic acid molecule into the cell(e.g., a liposome, protein coating, etc.).

Unless specifically indicated or implied, the terms “a,” “an,” and “the”signify “at least one,” as used herein.

Unless otherwise specifically explained, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this disclosure belongs.Definitions of common terms in molecular biology can be found in, forexample, Lewin B., Genes V, Oxford University Press, 1994 (ISBN0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and MeyersR. A. (ed.), Molecular Biology and Biotechnology: A Comprehensive DeskReference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted. All temperatures are in degrees Celsius.

IV. Nucleic Acid Molecules Comprising a Synthetic Brassica-DerivedCTP-Encoding Sequence

In some embodiments, this disclosure provides a nucleic acid moleculecomprising at least one nucleotide sequence encoding a syntheticBrassica-derived CTP operably linked to a nucleotide sequence ofinterest. In particular embodiments, the nucleotide sequence of interestmay be a nucleotide sequence that encodes a polypeptide of interest. Inparticular examples, a single nucleic acid molecule is provided thatencodes a polypeptide wherein a TraP8 or TraP9 sequence is fused to theN-terminus of a polypeptide of interest.

A synthetic Brassica-derived CTP may be derived from a Brassica EPSPSgene. In particular examples of such embodiments, the Brassica EPSPSgene may be one that comprises the nucleic acid sequence set forth asSEQ ID NO:14, or a homologous nucleic acid sequence from a differentEPSPS gene, or may be an ortholog of the Brassica EPSPS gene comprisingthe nucleic acid sequence set forth as SEQ ID NO:14 (for example, theBrassica EPSPS gene comprising the nucleic acid sequence set forth asSEQ ID NO:15).

In some embodiments, a synthetic Brassica-derived chloroplast transitpeptide may be a chimeric Brassica-derived CTP. A synthetic chimericBrassica-derived CTP may be derived from a reference Brassica CTPsequence by joining a first contiguous amino acid sequence comprisedwithin the reference Brassica CTP sequence to a second contiguous aminoacid sequence comprised within a different CTP sequence (e.g., a secondBrassica CTP sequence). In particular embodiments, the different CTPsequence comprising the second contiguous amino acid sequence may beencoded by a homologous gene sequence from a genome other than that ofthe Brassica sp. from which the reference sequence was obtained (e.g., adifferent Brassica sp., a plant other than a Brassica sp.; a lowerphotosynthetic eukaryote, for example, a Chlorophyte; and a prokaryote,for example, a Cyanobacterium or Agrobacterium). Thus, a nucleotidesequence encoding a synthetic Brassica-derived CTP may be derived from areference Brassica CTP-encoding gene sequence by fusing a nucleotidesequence that encodes a contiguous amino acid sequence of the referenceBrassica CTP sequence with a nucleotide sequence that encodes thecontiguous amino acid sequence from a different CTP sequence that ishomologous to the remainder of the reference Brassica CTP sequence. Inthese and other examples, the contiguous amino acid sequence of thereference Brassica CTP sequence may be located at the 5′ end or the 3′end of the synthetic Brassica-derived CTP.

In some embodiments, a synthetic chimeric Brassica-derived CTP may bederived from a plurality of Brassica CTP sequences (including areference Brassica CTP sequence) by joining a contiguous amino acidsequence comprised within one Brassica CTP sequence to a contiguousamino acid sequence comprised within a different Brassica CTP sequence.In particular embodiments, the plurality of Brassica CTP sequences maybe encoded by orthologous gene sequences in different Brassica species.In some examples, the plurality of Brassica CTP sequences may be exactlytwo Brassica CTP sequences. Thus, a nucleotide sequence encoding asynthetic chimeric Brassica-derived CTP may be derived from twohomologous (e.g., substantially homologous) Brassica CTP-encoding genesequences (e.g., orthologous gene sequences) by fusing the nucleotidesequence that encodes a contiguous amino acid sequence of one of theBrassica CTP sequences with the nucleotide sequence that encodes thecontiguous amino acid sequence from the other of the Brassica CTPsequences that is homologous to the remainder of the first Brassica CTPsequence. TraP8 and TraP9 are illustrative examples of such a syntheticchimeric Brassica-derived CTP.

One of ordinary skill in the art will understand that, following theselection of a first contiguous amino acid sequence within a referenceBrassica CTP sequence, the identification and selection of thecontiguous amino acid sequence from the remainder of a homologous CTPsequence according to the foregoing derivation process is unambiguousand automatic. In some examples, the first contiguous amino acidsequence may be between about 25 and about 41 amino acids in length(e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, and 42 amino acids in length). In some embodiments, the firstcontiguous amino acid sequence within the reference Brassica CTPsequence is defined by the position at the 3′ end of a “SVSL” (SEQ IDNO:13) motif that is conserved within some Brassica EPSPS genes.

Examples of synthetic chimeric Brassica-derived CTP sequences accordingto the foregoing process are represented by SEQ ID NO:3 and SEQ ID NO:4.In view of the degeneracy of the genetic code, the genus of nucleotidesequences encoding these peptides will be immediately envisioned by oneof ordinary skill in the art. Examples of such polynucleotide sequencesinclude SEQ ID NOs: 5, 6, 8, and 9. These particular examples illustratethe structural features of synthetic chimeric Brassica-derived CTPs byincorporating contiguous sequences from a homologous CTP from one ofseveral ESPSP orthologs of a B. napus ESPSP gene.

Some embodiments include functional variants of a syntheticBrassica-derived chloroplast transit peptide, and/or nucleic acidsencoding the same. Such functional variants include, for example andwithout limitation: a synthetic Brassica-derived CTP-encoding sequencethat is derived from a homolog and/or ortholog of one or both of theBrassica CTP-encoding sequences set forth as SEQ ID NOs:14 and/or SEQ IDNO:15, and/or a CTP encoded thereby; a nucleic acid that encodes asynthetic Brassica-derived CTP that comprises a contiguous amino acidsequence within SEQ ID NO:1 and/or SEQ ID NO:2, and/or a CTP encodedthereby; a truncated synthetic Brassica-derived CTP-encoding sequencethat comprises a contiguous nucleic acid sequence within one of SEQ IDNOs:5, 6, 8, and 9; a truncated synthetic Brassica-derived CTP-encodingsequence that comprises a contiguous nucleic acid sequence that issubstantially homologous to one of SEQ ID NOs: 5, 6, 8, and 9; atruncated synthetic Brassica-derived CTP that comprises a contiguousamino acid sequence within one of SEQ ID NOs: 3 and 4; a nucleic acidthat encodes a synthetic Brassica-derived CTP comprising a contiguousamino acid sequence within one of SEQ ID NOs: 5, 6, 8, and 9, and/or aCTP encoded thereby; a nucleic acid that encodes a syntheticBrassica-derived CTP comprising a contiguous amino acid sequence withinone of SEQ ID NOs: 3 and 4 that has one or more conservative amino acidsubstitutions, and/or a CTP encoded thereby; and a nucleic acid thatencodes a synthetic Brassica-derived CTP comprising a contiguous aminoacid sequence within one of SEQ ID NOs: 3 and 4 that has one or morenon-conservative amino acid substitutions that are demonstrated todirect an operably linked peptide to a plastid in a plastid-containingcell, and/or a CTP encoded thereby.

Thus, some embodiments of the invention include a nucleic acid moleculecomprising a nucleotide sequence encoding a synthetic chimericBrassica-derived CTP comprising one or more conservative amino acidsubstitutions. Such a nucleic acid molecule may be useful, for example,in facilitating manipulation of a CTP-encoding sequence of the inventionin molecular biology techniques. For example, in some embodiments, aCTP-encoding sequence of the invention may be introduced into a suitablevector for sub-cloning of the sequence into an expression vector, or aCTP-encoding sequence of the invention may be introduced into a nucleicacid molecule that facilitates the production of a further nucleic acidmolecule comprising the CTP-encoding sequence operably linked to anucleotide sequence of interest. In these and further embodiments, oneor more amino acid positions in the sequence of a synthetic chimericBrassica-derived CTP may be deleted. For example, the sequence of asynthetic chimeric Brassica-derived CTP may be modified such that theamino acid(s) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 positions in the sequence are deleted. An alignment ofhomologous CTP sequences may be used to provide guidance as to whichamino acids may be deleted without affecting the function of thesynthetic CTP.

In particular examples, a synthetic Brassica-derived chloroplast transitpeptide is less than 80 amino acids in length. For example, a syntheticBrassica-derived CTP may be 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69,68, 67, 66, 65, 64, 63, 62, 61, 60, or fewer amino acids in length. Incertain examples, a synthetic Brassica-derived CTP may be about 65,about 68, about 72, or about 74 amino acids in length. In these andfurther examples, a synthetic Brassica-derived CTP may comprise an aminoacid sequence set forth in one of SEQ ID NOs: 3 and 4, or a functionalvariant of any of the foregoing. Thus, a synthetic Brassica-derived CTPmay comprise an amino acid sequence comprising one of SEQ ID NOs: 3 and4 or a functional variant thereof, wherein the length of the syntheticBrassica-derived CTP is less than 80 amino acids in length. In certainexamples, a synthetic Brassica-derived CTP may comprise an amino acidsequence that is, e.g., at least 80%, at least 85%, at least 90%, atleast 92%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to one of SEQ ID NOs: 3 and4.

All of the nucleotide sequences that encode a particular syntheticBrassica-derived CTP, for example, the TraP8 peptide of SEQ ID NO:3 andthe TraP9 peptide of SEQ ID NO:4, or functional variants of any of theforegoing including any specific deletions and/or conservative aminoacid substitutions, will be recognizable by those of skill in the art inview of the present disclosure. The degeneracy of the genetic codeprovides a finite number of coding sequences for a particular amino acidsequence. The selection of a particular sequence to encode a syntheticBrassica-derived CTP is within the discretion of the practitioner.Different coding sequences may be desirable in different applications.For example, to increase expression of the synthetic Brassica-derivedCTP in a particular host, a coding sequence may be selected thatreflects the codon usage bias of the host. By way of example, asynthetic Brassica-derived CTP may be encoded by a nucleotide sequenceset forth as one of SEQ ID NOs: 5, 6, 8, and 9.

In nucleic acid molecules provided in some embodiments of the invention,the last codon of a nucleotide sequence encoding a syntheticBrassica-derived CTP and the first codon of a nucleotide sequence ofinterest may be separated by any number of nucleotide triplets, e.g.,without coding for an intron or a “STOP.” In some examples, a sequenceencoding the first amino acids of a mature protein normally associatedwith a chloroplast transit peptide in a natural precursor polypeptidemay be present between the last codon of a nucleotide sequence encodinga synthetic Brassica-derived CTP and the first codon of a nucleotidesequence of interest. A sequence separating a nucleotide sequenceencoding a synthetic Brassica-derived CTP and the first codon of anucleotide sequence of interest may, for example, consist of anysequence, such that the amino acid sequence encoded is not likely tosignificantly alter the translation of the chimeric polypeptide and itstranslocation to a plastid. In these and further embodiments, the lastcodon of a nucleotide sequence encoding a synthetic Brassica-derivedchloroplast transit peptide may be fused in phase-register with thefirst codon of the nucleotide sequence of interest directly contiguousthereto, or separated therefrom by no more than a short peptidesequence, such as that encoded by a synthetic nucleotide linker (e.g., anucleotide linker that may have been used to achieve the fusion).

In some embodiments, it may be desirable to modify the nucleotides of anucleotide sequence of interest and/or a synthetic Brassica-derivedCTP-encoding sequence fused thereto in a single coding sequence, forexample, to enhance expression of the coding sequence in a particularhost. The genetic code is redundant with 64 possible codons, but mostorganisms preferentially use a subset of these codons. The codons thatare utilized most often in a species are called optimal codons, andthose not utilized very often are classified as rare or low-usagecodons. Zhang et al. (1991), Gene 105:61-72. Codons may be substitutedto reflect the preferred codon usage of a particular host in a processsometimes referred to as “codon optimization.” Optimized codingsequences containing codons preferred by a particular prokaryotic oreukaryotic host may be prepared, for example, to increase the rate oftranslation or to produce recombinant RNA transcripts having desirableproperties (e.g., a longer half-life, as compared with transcriptsproduced from a non-optimized sequence).

Any polypeptide may be targeted to a plastid of a plastid-containingcell by incorporation of a synthetic Brassica-derived CTP sequence. Forexample, a polypeptide may be linked to a synthetic Brassica-derived CTPsequence in some embodiments, so as to direct the polypeptide to aplastid in a cell wherein the linked polypeptide-CTP molecule isexpressed. In particular embodiments, a polypeptide targeted to aplastid by incorporation of a synthetic Brassica-derived CTP sequencemay be, for example, a polypeptide that is normally expressed in aplastid of a cell wherein the polypeptide is natively expressed. Forexample and without limitation, a polypeptide targeted to a plastid byincorporation of a synthetic Brassica-derived CTP sequence may be apolypeptide involved in herbicide resistance, virus resistance,bacterial pathogen resistance, insect resistance, nematode resistance,or fungal resistance. See, e.g., U.S. Pat. Nos. 5,569,823; 5,304,730;5,495,071; 6,329,504; and 6,337,431. A polypeptide targeted to a plastidby incorporation of a synthetic Brassica-derived CTP sequence mayalternatively be, for example and without limitation, a polypeptideinvolved in plant vigor or yield (including polypeptides involved intolerance for extreme temperatures, soil conditions, light levels, waterlevels, and chemical environment), or a polypeptide that may be used asa marker to identify a plant comprising a trait of interest (e.g., aselectable marker gene product, a polypeptide involved in seed color,etc.).

Non-limiting examples of polypeptides involved in herbicide resistancethat may be linked to a synthetic Brassica-derived CTP sequence in someembodiments of the invention include: acetolactase synthase (ALS),mutated ALS, and precursors of ALS (see, e.g., U.S. Pat. No. 5,013,659);EPSPS (see, e.g., U.S. Pat. Nos. 4,971,908 and 6,225,114), such as a CP4EPSPS, a class III EPSPS, or a class IV EPSPS; enzymes that modify aphysiological process that occurs in a plastid, includingphotosynthesis, and synthesis of fatty acids, amino acids, oils,arotenoids, terpenoids, starch, etc. Other non-limiting examples ofpolypeptides that may be linked to a synthetic Brassica-derivedchloroplast transit peptide in particular embodiments include:zeaxanthin epoxidase, choline monooxygenase, ferrochelatase, omega-3fatty acid desaturase, glutamine synthetase, starch modifying enzymes,polypeptides involved in synthesis of essential amino acids, provitaminA, hormones, Bt toxin proteins, etc. Nucleotide sequences encoding theaforementioned peptides are known in the art, and such nucleotidesequences may be operably linked to a nucleotide sequence encoding asynthetic Brassica-derived CTP to be expressed into a polypeptidecomprising the polypeptide of interest linked to the syntheticBrassica-derived CTP. Furthermore, additional nucleotide sequencesencoding any of the aforementioned polypeptides may be identified bythose of skill in the art (for example, by cloning of genes with highhomology to other genes encoding the particular polypeptide). Once sucha nucleotide sequence has been identified, it is a straightforwardprocess to design a nucleotide sequence comprising a syntheticBrassica-derived CTP-encoding sequence operably linked to the identifiednucleotide sequence, or a sequence encoding an equivalent polypeptide.

V. Expression of Polypeptides Comprising a Synthetic Brassica-DerivedChloroplast Transit Peptide

In some embodiments, at least one nucleic acid molecule(s) comprising anucleotide sequence encoding a polypeptide comprising at least onesynthetic Brassica-derived CTP, or functional equivalent thereof, may beintroduced into a cell, tissue, or organism for expression of thepolypeptide therein. In particular embodiments, a nucleic acid moleculemay comprise a nucleotide sequence of interest operably linked to anucleotide sequence encoding a synthetic Brassica-derived CTP. Forexample, a nucleic acid molecule may comprise a coding sequence encodinga polypeptide comprising at least one synthetic Brassica-derived CTP andat least an additional peptide sequence encoded by a nucleotide sequenceof interest. In some embodiments, a nucleic acid molecule of theinvention may be introduced into a plastid-containing host cell, tissue,or organism (e.g., a plant cell, plant tissue, and plant), such that apolypeptide may be expressed from the nucleic acid molecule in theplastid-containing host cell, tissue, or organism, wherein the expressedpolypeptide comprises at least one synthetic Brassica-derived CTP and atleast an additional peptide sequence encoded by a nucleotide sequence ofinterest. In certain examples, the synthetic Brassica-derived CTP ofsuch an expressed polypeptide may facilitate targeting of a portion ofthe polypeptide comprising at least the additional peptide sequence to aplastid of the host cell, tissue, or organism.

In some embodiments, a nucleic acid molecule of the invention may beintroduced into a plastid-containing cell by one of any of themethodologies known to those of skill in the art. In particularembodiments, a host cell, tissue, or organism may be contacted with anucleic acid molecule of the invention in order to introduce the nucleicacid molecule into the cell, tissue, or organism. In particularembodiments, a cell may be transformed with a nucleic acid molecule ofthe invention such that the nucleic acid molecule is introduced into thecell, and the nucleic acid molecule is stably integrated into the genomeof the cell. In some embodiments, a nucleic acid molecule comprising atleast one nucleotide sequence encoding a synthetic Brassica-derived CTPoperably linked to a nucleotide sequence of interest may be used fortransformation of a cell, for example, a plastid-containing cell (e.g.,a plant cell). In order to initiate or enhance expression, a nucleicacid molecule may comprise one or more regulatory sequences, whichregulatory sequences may be operably linked to the nucleotide sequenceencoding a polypeptide comprising at least one syntheticBrassica-derived CTP.

A nucleic acid molecule may, for example, be a vector system including,for example, a linear or a closed circular plasmid. In particularembodiments, the vector may be an expression vector. Nucleic acidsequences of the invention may, for example, be inserted into a vector,such that the nucleic acid sequence is operably linked to one or moreregulatory sequences. Many vectors are available for this purpose, andselection of the particular vector may depend, for example, on the sizeof the nucleic acid to be inserted into the vector and the particularhost cell to be transformed with the vector. A vector typically containsvarious components, the identity of which depend on a function of thevector (e.g., amplification of DNA and expression of DNA), and theparticular host cell(s) with which the vector is compatible.

Some embodiments may include a plant transformation vector thatcomprises a nucleotide sequence comprising at least one of theabove-described regulatory sequences operatively linked to one or morenucleotide sequence(s) encoding a polypeptide comprising at least onesynthetic Brassica-derived CTP. The one or more nucleotide sequences maybe expressed, under the control of the regulatory sequence(s), in aplant cell, tissue, or organism to produce a polypeptide comprising asynthetic Brassica-derived CTP that targets at least a portion of thepolypeptide to a plastid of the plant cell, tissue, or organism.

In some embodiments, a regulatory sequence operably linked to anucleotide sequence encoding a polypeptide comprising at least onesynthetic Brassica-derived CTP, may be a promoter sequence thatfunctions in a host cell, such as a bacterial cell wherein the nucleicacid molecule is to be amplified, or a plant cell wherein the nucleicacid molecule is to be expressed. Promoters suitable for use in nucleicacid molecules of the invention include those that are inducible, viral,synthetic, or constitutive, all of which are well known in the art.Non-limiting examples of promoters that may be useful in embodiments ofthe invention are provided by: U.S. Pat. No. 6,437,217 (maize RS81promoter); U.S. Pat. No. 5,641,876 (rice actin promoter); U.S. Pat. No.6,426,446 (maize RS324 promoter); U.S. Pat. No. 6,429,362 (maize PR-1promoter); U.S. Pat. No. 6,232,526 (maize A3 promoter); U.S. Pat. No.6,177,611 (constitutive maize promoters); U.S. Pat. Nos. 5,322,938,5,352,605, 5,359,142, and 5,530,196 (35S promoter); U.S. Pat. No.6,433,252 (maize L3 oleosin promoter); U.S. Pat. No. 6,429,357 (riceactin 2 promoter, and rice actin 2 intron); U.S. Pat. No. 6,294,714(light-inducible promoters); U.S. Pat. No. 6,140,078 (salt-induciblepromoters); U.S. Pat. No. 6,252,138 (pathogen-inducible promoters); U.S.Pat. No. 6,175,060 (phosphorous deficiency-inducible promoters); U.S.Pat. No. 6,388,170 (bidirectional promoters); U.S. Pat. No. 6,635,806(gamma-coixin promoter); and U.S. patent application Ser. No. 09/757,089(maize chloroplast aldolase promoter).

Additional exemplary promoters include the nopaline synthase (NOS)promoter (Ebert et al. (1987), Proc. Natl. Acad. Sci. USA84(16):5745-9); the octopine synthase (OCS) promoter (which is carriedon tumor-inducing plasmids of Agrobacterium tumefaciens); thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Spromoter (Lawton et al. (1987), Plant Mol. Biol. 9:315-24); the CaMV 35Spromoter (Odell et al. (1985), Nature 313:810-2; the figwort mosaicvirus 35S-promoter (Walker et al. (1987), Proc. Natl. Acad. Sci. USA84(19):6624-8); the sucrose synthase promoter (Yang and Russell (1990),Proc. Natl. Acad. Sci. USA 87:4144-8); the R gene complex promoter(Chandler et al. (1989), Plant Cell 1:1175-83); the chlorophyll a/bbinding protein gene promoter; CaMV35S (U.S. Pat. Nos. 5,322,938,5,352,605, 5,359,142, and 5,530,196); FMV35S (U.S. Pat. Nos. 6,051,753,and 5,378,619); a PC1SV promoter (U.S. Pat. No. 5,850,019); the SCP1promoter (U.S. Pat. No. 6,677,503); and AGRtu.nos promoters (GenBankAccession No. V00087; Depicker et al. (1982), J. Mol. Appl. Genet.1:561-73; Bevan et al. (1983), Nature 304:184-7).

In particular embodiments, nucleic acid molecules of the invention maycomprise a tissue-specific promoter. A tissue-specific promoter is anucleotide sequence that directs a higher level of transcription of anoperably linked nucleotide sequence in the tissue for which the promoteris specific, relative to the other tissues of the organism. Examples oftissue-specific promoters include, without limitation: tapetum-specificpromoters; anther-specific promoters; pollen-specific promoters (See,e.g., U.S. Pat. No. 7,141,424, and International PCT Publication No. WO99/042587); ovule-specific promoters; (See, e.g., U.S. PatentApplication No. 2001/047525 A1); fruit-specific promoters (See, e.g.,U.S. Pat. Nos. 4,943,674, and 5,753,475); and seed-specific promoters(See, e.g., U.S. Pat. Nos. 5,420,034, and 5,608,152). In someembodiments, a developmental stage-specific promoter (e.g., a promoteractive at a later stage in development) may be used in a composition ormethod of the invention.

Additional regulatory sequences that may in some embodiments be operablylinked to a nucleic acid molecule include 5′ UTRs located between apromoter sequence and a coding sequence that function as a translationleader sequence. The translation leader sequence is present in thefully-processed mRNA, and it may affect processing of the primarytranscript, and/or RNA stability. Examples of translation leadersequences include maize and petunia heat shock protein leaders (U.S.Pat. No. 5,362,865), plant virus coat protein leaders, plant rubiscoleaders, and others. See, e.g., Turner and Foster (1995), MolecularBiotech. 3(3):225-36. Non-limiting examples of 5′ UTRs are provided by:GmHsp (U.S. Pat. No. 5,659,122); PhDnaK (U.S. Pat. No. 5,362,865);AtAnt1; TEV (Carrington and Freed (1990), J. Virol. 64:1590-7); andAGRtunos (GenBank Accession No. V00087; and Bevan et al. (1983), Nature304:184-7).

Additional regulatory sequences that may in some embodiments be operablylinked to a nucleic acid molecule also include 3′ non-translatedsequences, 3′ transcription termination regions, or poly-adenylationregions. These are genetic elements located downstream of a nucleotidesequence, and include polynucleotides that provide polyadenylationsignal, and/or other regulatory signals capable of affectingtranscription or mRNA processing. The polyadenylation signal functionsin plants to cause the addition of polyadenylate nucleotides to the 3′end of the mRNA precursor. The polyadenylation sequence can be derivedfrom a variety of plant genes, or from T-DNA genes. A non-limitingexample of a 3′ transcription termination region is the nopalinesynthase 3′ region (nos 3; Fraley et al. (1983), Proc. Natl. Acad. Sci.USA 80:4803-7). An example of the use of different 3′ nontranslatedregions is provided in Ingelbrecht et al., (1989), Plant Cell 1:671-80.Non-limiting examples of polyadenylation signals include one from aPisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al. (1984), EMBO J.3:1671-9) and AGRtu.nos (GenBank Accession No. E01312).

A recombinant nucleic acid molecule or vector of the present inventionmay comprise a selectable marker that confers a selectable phenotype ona transformed cell, such as a plant cell. Selectable markers may also beused to select for plants or plant cells that comprise recombinantnucleic acid molecule of the invention. The marker may encode biocideresistance, antibiotic resistance (e.g., kanamycin, Geneticin (G418),bleomycin, hygromycin, etc.), or herbicide resistance (e.g., glyphosate,etc.). Examples of selectable markers include, but are not limited to: aneo gene which codes for kanamycin resistance and can be selected forusing kanamycin, G418, etc.; a pat or bar gene which codes for bialaphosresistance; a mutant EPSP synthase gene which encodes glyphosateresistance; a nitrilase gene which confers resistance to bromoxynil; amutant acetolactate synthase gene (ALS) which confers imidazolinone orsulfonylurea resistance; and a methotrexate resistant DHFR gene.Multiple selectable markers are available that confer resistance toampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin,spectinomycin, rifampicin, streptomycin and tetracycline, and the like.Examples of such selectable markers are illustrated in, e.g., U.S. Pat.Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047.

A recombinant nucleic acid molecule or vector of the present inventionmay also or alternatively include a screenable marker. Screenablemarkers may be used to monitor expression. Exemplary screenable markersinclude a β-glucuronidase or uidA gene (GUS) which encodes an enzyme forwhich various chromogenic substrates are known (Jefferson et al. (1987),Plant Mol. Biol. Rep. 5:387-405); an R-locus gene, which encodes aproduct that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al. (1988), “Molecular cloning ofthe maize R-nj allele by transposon tagging with Ac.” In 18th StadlerGenetics Symposium, P. Gustafson and R. Appels, eds. (New York: Plenum),pp. 263-82); a β-lactamase gene (Sutcliffe et al. (1978), Proc. Natl.Acad. Sci. USA 75:3737-41); a gene which encodes an enzyme for whichvarious chromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a luciferase gene (Ow et al. (1986), Science 234:856-9);a xy/E gene that encodes a catechol dioxygenase that can convertchromogenic catechols (Zukowski et al. (1983), Gene 46(2-3):247-55); anamylase gene (Ikatu et al. (1990) Bio/Technol. 8:241-2); a tyrosinasegene which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone which in turn condenses to melanin (Katz et al. (1983), 1Gen. Microbiol. 129:2703-14); and an α-galactosidase.

Suitable methods for transformation of host cells include any method bywhich DNA can be introduced into a cell, for example and withoutlimitation: by transformation of protoplasts (see, e.g., U.S. Pat. No.5,508,184); by desiccation/inhibition-mediated DNA uptake (see, e.g.,Potrykus et al. (1985), Mol. Gen. Genet. 199:183-8); by electroporation(see, e.g., U.S. Pat. No. 5,384,253); by agitation with silicon carbidefibers (see, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765); byAgrobacterium-mediated transformation (see, e.g., U.S. Pat. Nos.5,563,055, 5,591,616, 5,693,512, 5,824,877, 5,981,840, and 6,384,301);and by acceleration of DNA-coated particles (see, e.g., U.S. Pat. Nos.5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865);etc. Through the application of techniques such as these, the cells ofvirtually any species may be stably transformed. In some embodiments,transforming DNA is integrated into the genome of the host cell. In thecase of multicellular species, transgenic cells may be regenerated intoa transgenic organism. Alternatively, the transgenic cells may not becapable of regeneration to a plant. Any of these techniques may be usedto produce a transgenic plant, for example, comprising one or morenucleic acid sequences of the invention in the genome of the transgenicplant.

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The T_(i) andR_(i) plasmids of A. tumefaciens and A. rhizogenes, respectively, carrygenes responsible for genetic transformation of the plant. The T_(i)(tumor-inducing)-plasmids contain a large segment, known as T-DNA, whichis transferred to transformed plants. Another segment of the T_(i)plasmid, the vir region, is responsible for T-DNA transfer. The T-DNAregion is bordered by terminal repeats. In some modified binary vectors,the tumor-inducing genes have been deleted, and the functions of the virregion are utilized to transfer foreign DNA bordered by the T-DNA bordersequences. The T-region may also contain, for example, a selectablemarker for efficient recovery of transgenic plants and cells, and amultiple cloning site for inserting sequences for transfer such as asynthetic Brassica-derived CTP-encoding nucleic acid.

Thus, in some embodiments, a plant transformation vector may be derivedfrom a T_(i) plasmid of A. tumefaciens (See, e.g., U.S. Pat. Nos.4,536,475, 4,693,977, 4,886,937, and 5,501,967; and European Patent EP 0122 791) or a Ri plasmid of A. rhizogenes. Additional planttransformation vectors include, for example and without limitation,those described by Herrera-Estrella et al. (1983), Nature 303:209-13;Bevan et al. (1983), Nature 304:184-7; Klee et al. (1985), Bio/Technol.3:637-42; and in European Patent EP 0 120 516, and those derived fromany of the foregoing. Other bacteria such as Sinorhizobium, Rhizobium,and Mesorhizobium that interact with plants naturally can be modified tomediate gene transfer to a number of diverse plants. Theseplant-associated symbiotic bacteria can be made competent for genetransfer by acquisition of both a disarmed T_(i) plasmid and a suitablebinary vector.

After providing exogenous DNA to recipient cells, transformed cells aregenerally identified for further culturing and plant regeneration. Inorder to improve the ability to identify transformed cells, one maydesire to employ a selectable or screenable marker gene, as previouslyset forth, with the vector used to generate the transformant. In thecase where a selectable marker is used, transformed cells are identifiedwithin the potentially transformed cell population by exposing the cellsto a selective agent or agents. In the case where a screenable marker isused, cells may be screened for the desired marker gene trait.

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In some embodiments, any suitableplant tissue culture media (e.g., MS and N6 media) may be modified byincluding further substances, such as growth regulators. Tissue may bemaintained on a basic media with growth regulators until sufficienttissue is available to begin plant regeneration efforts, or followingrepeated rounds of manual selection, until the morphology of the tissueis suitable for regeneration (e.g., at least 2 weeks), then transferredto media conducive to shoot formation. Cultures are transferredperiodically until sufficient shoot formation has occurred. Once shootsare formed, they are transferred to media conducive to root formation.Once sufficient roots are formed, plants can be transferred to soil forfurther growth and maturity.

To confirm the presence of a nucleic acid molecule of interest (forexample, a nucleotide sequence encoding a polypeptide comprising atleast one synthetic Brassica-derived CTP) in a regenerating plant, avariety of assays may be performed. Such assays include, for example:molecular biological assays, such as Southern and Northern blotting,PCR, and nucleic acid sequencing; biochemical assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAand/or Western blots) or by enzymatic function; plant part assays, suchas leaf or root assays; and analysis of the phenotype of the wholeregenerated plant.

By way of example, integration events may be analyzed by PCRamplification using, e.g., oligonucleotide primers specific for anucleotide sequence of interest. PCR genotyping is understood toinclude, but not be limited to, polymerase-chain reaction (PCR)amplification of genomic DNA derived from isolated host plant tissuepredicted to contain a nucleic acid molecule of interest integrated intothe genome, followed by standard cloning and sequence analysis of PCRamplification products. Methods of PCR genotyping have been welldescribed (see, e.g., Rios, G. et al. (2002), Plant J. 32:243-53), andmay be applied to genomic DNA derived from any plant species (e.g., Z.mays or G. max) or tissue type, including cell cultures.

A transgenic plant formed using Agrobacterium-dependent transformationmethods typically contains a single recombinant DNA sequence insertedinto one chromosome. The single recombinant DNA sequence is referred toas a “transgenic event” or “integration event.” Such transgenic plantsare heterozygous for the inserted DNA sequence. In some embodiments, atransgenic plant homozygous with respect to a transgene may be obtainedby sexually mating (selfing) an independent segregant transgenic plantthat contains a single exogenous gene sequence to itself, for example,an F₀ plant, to produce F₁ seed. One fourth of the F₁ seed produced willbe homozygous with respect to the transgene. Germinating F₁ seed resultsin plants that can be tested for heterozygosity, typically using a SNPassay or a thermal amplification assay that allows for the distinctionbetween heterozygotes and homozygotes (i.e., a zygosity assay).

In particular embodiments, copies of at least one polypeptide comprisingat least one synthetic Brassica-derived CTP are produced in aplastid-containing cell, into which has been introduced at least onenucleic acid molecule(s) comprising a nucleotide sequence encoding theat least one polypeptide comprising at least one syntheticBrassica-derived CTP. Each polypeptide comprising at least one syntheticBrassica-derived CTP may be expressed from multiple nucleic acidsequences introduced in different transformation events, or from asingle nucleic acid sequence introduced in a single transformationevent. In some embodiments, a plurality of such polypeptides isexpressed under the control of a single promoter. In other embodiments,a plurality of such polypeptides is expressed under the control ofmultiple promoters. Single polypeptides may be expressed that comprisemultiple peptide sequences, each of which peptide sequences is to betargeted to a plastid.

In addition to direct transformation of a plant with a recombinantnucleic acid molecule, transgenic plants can be prepared by crossing afirst plant having at least one transgenic event with a second plantlacking such an event. For example, a recombinant nucleic acid moleculecomprising a nucleotide sequence encoding a polypeptide comprising atleast one synthetic Brassica-derived CTP may be introduced into a firstplant line that is amenable to transformation, to produce a transgenicplant, which transgenic plant may be crossed with a second plant line tointrogress the nucleotide sequence that encodes the polypeptide into thesecond plant line.

VI. Plant Materials Comprising a Synthetic Brassica-Derived ChloroplastTransit Peptide-Directed Polypeptide

In some embodiments, a plant cell is provided, wherein the plant cellcomprises a nucleotide sequence encoding a polypeptide comprising atleast one synthetic Brassica-derived CTP. In particular embodiments,such a plant cell may be produced by transformation of a plant cell thatis not capable of regeneration to produce a plant. In some embodiments,a plant is provided, wherein the plant comprises a plant cell comprisinga nucleotide sequence encoding a polypeptide comprising at least onesynthetic Brassica-derived CTP. In particular embodiments, such a plantmay be produced by transformation of a plant tissue or plant cell, andregeneration of a whole plant. In further embodiments, such a plant maybe obtained from a commercial source, or through introgression of anucleic acid comprising a nucleotide sequence encoding a polypeptidecomprising at least one synthetic Brassica-derived CTP into a germplasm.In particular embodiments, such a plant comprises plant cells comprisinga nucleotide sequence encoding a polypeptide comprising at least onesynthetic Brassica-derived CTP that are not capable of regeneration toproduce a plant. Plant materials comprising a plant cell comprising anucleotide sequence encoding a polypeptide comprising at least onesynthetic Brassica-derived CTP are also provided. Such a plant materialmay be obtained from a plant comprising the plant cell.

A transgenic plant, nonregenerable plant cell, or plant materialcomprising a nucleotide sequence encoding a polypeptide comprising atleast one synthetic Brassica-derived CTP may in some embodiments exhibitone or more of the following characteristics: expression of thepolypeptide in a cell of the plant; expression of a portion of thepolypeptide in a plastid of a cell of the plant; import of thepolypeptide from the cytosol of a cell of the plant into a plastid ofthe cell; plastid-specific expression of the polypeptide in a cell ofthe plant; and/or localization of the polypeptide in a cell of theplant. Such a plant may additionally have one or more desirable traitsother than expression of the encoded polypeptide. Such traits mayinclude, for example: resistance to insects, other pests, anddisease-causing agents; tolerances to herbicides; enhanced stability,yield, or shelf-life; environmental tolerances; pharmaceuticalproduction; industrial product production; and nutritional enhancements.

A transgenic plant according to the invention may be any plant capableof being transformed with a nucleic acid molecule of the invention.Accordingly, the plant may be a dicot or monocot. Non-limiting examplesof dicotyledonous plants usable in the present methods includeArabidopsis, alfalfa, beans, broccoli, cabbage, carrot, cauliflower,celery, Chinese cabbage, cotton, cucumber, eggplant, lettuce, melon,pea, pepper, peanut, potato, pumpkin, radish, rapeseed, spinach,soybean, squash, sugarbeet, sunflower, tobacco, tomato, and watermelon.Non-limiting examples of monocotyledonous plants usable in the presentmethods include corn, Brassica, onion, rice, sorghum, wheat, rye,millet, sugarcane, oat, triticale, switchgrass, and turfgrass.Transgenic plants according to the invention may be used or cultivatedin any manner.

Some embodiments also provide commodity products containing one or morenucleotide sequences encoding a polypeptide comprising at least onesynthetic Brassica-derived CTP, for example, a commodity productproduced from a recombinant plant or seed containing one or more of suchnucleotide sequences. Commodity products containing one or morenucleotide sequences encoding a polypeptide comprising at least onesynthetic Brassica-derived CTP include, for example and withoutlimitation: food products, meals, oils, or crushed or whole grains orseeds of a plant comprising one or more nucleotide sequences encoding apolypeptide comprising at least one synthetic Brassica-derived CTP. Thedetection of one or more nucleotide sequences encoding a polypeptidecomprising at least one synthetic Brassica-derived CTP in one or morecommodity or commodity products is de facto evidence that the commodityor commodity product was at least in part produced from a plantcomprising one or more nucleotide sequences encoding a polypeptidecomprising at least one synthetic Brassica-derived CTP. In particularembodiments, a commodity product of the invention comprise a detectableamount of a nucleic acid sequence encoding a polypeptide comprising atleast one synthetic Brassica-derived CTP. In some embodiments, suchcommodity products may be produced, for example, by obtaining transgenicplants and preparing food or feed from them.

In some embodiments, a transgenic plant, nonregenerable plant cell, orseed comprising a transgene comprising a nucleotide sequence encoding apolypeptide comprising at least one synthetic Brassica-derived CTP alsomay comprise at least one other transgenic event in its genome,including without limitation: a transgenic event from which istranscribed an iRNA molecule; a gene encoding an insecticidal protein(e.g., an Bacillus thuringiensis insecticidal protein); an herbicidetolerance gene (e.g., a gene providing tolerance to glyphosate); and agene contributing to a desirable phenotype in the transgenic plant(e.g., increased yield, altered fatty acid metabolism, or restoration ofcytoplasmic male sterility).

VII. Synthetic Brassica-Derived Chloroplast Transit Peptide-MediatedLocalization of Gene Products to Plastids

Some embodiments of the present invention provide a method forexpression and/or localization of a gene product to a plastid (e.g., achloroplast). In particular embodiments, the gene product may be amarker gene product, for example, a fluorescent molecule. Expression ofthe gene product as part of a polypeptide also comprising a syntheticBrassica-derived CTP may provide a system to evaluate theplastid-localizing capabilities of a particular syntheticBrassica-derived CTP sequence. In some embodiments, expression of amarker gene product as part of a synthetic Brassica-derivedCTP-containing polypeptide is utilized to target expression of themarker gene product to a plastid of a cell wherein the polypeptide isexpressed. In certain embodiments, such a marker gene product islocalized in plastid(s) of the host cell. For example, the marker geneproduct may be expressed at higher levels in the plastid(s) than in thecytosol or other organelles of the host cell; the marker gene productmay be expressed at much higher levels in the plastid(s); the markergene product may be expressed essentially only in the plastid(s); or themarker gene product may be expressed entirely in the plastid(s), suchthat expression in the cytosol or non-plastid organelles cannot bedetected.

In some embodiments, a polypeptide comprising a functional variant of asynthetic Brassica-derived CTP, wherein the polypeptide is operablylinked to a marker gene product is used to evaluate the characteristicsof the functional variant peptide. For example, the sequence of asynthetic Brassica-derived CTP may be varied, e.g., by introducing atleast one conservative mutation(s) into the synthetic Brassica-derivedCTP, and the resulting variant peptide may be linked to a marker geneproduct. After expression in a suitable host cell (for example, a cellwherein one or more regulatory elements in the expression construct areoperable), expression of the marker gene product may be determined. Bycomparing the sub-cellular localization of the marker gene productbetween the reference synthetic Brassica-derived CTP-marker constructand the variant peptide-marker construct, it may be determined whetherthe variant peptide provides, for example, greater plastid localization,or substantially identical plastid localization. Such a variant may beconsidered a functional variant. By identifying functional variants ofsynthetic Brassica-derived CTP that provide greater plasticlocalization, the mutations in such variants may be incorporated intofurther variants of synthetic Brassica-derived CTPs. Performing multiplerounds of this evaluation process, and subsequently incorporatingidentified favorable mutations in a synthetic Brassica-derived CTPsequence, may yield an iterative process for optimization of a syntheticBrassica-derived CTP sequence. Such optimized synthetic Brassica-derivedCTP sequences, and nucleotide sequences encoding the same, areconsidered part of the present invention, whether or not such optimizedsynthetic Brassica-derived CTP sequences may be further optimized byadditional mutation.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to theextent they are not inconsistent with the explicit details of thisdisclosure, and are so incorporated to the same extent as if eachreference were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

The following Examples are provided to illustrate certain particularfeatures and/or aspects. These Examples should not be construed to limitthe disclosure to the particular features or aspects described.

EXAMPLES Example 1: Design and Production of Chimeric ChloroplastTransit Peptide (TraP) Sequences

Plastids are cytoplasmic organelles found in higher plant species andare present in all plant tissues. Choloroplasts are a specific type ofplastid found in green photosynthetic tissues which are responsible foressential physiological functions. For example, one such primaryphysiological function is the synthesis of aromatic amino acids requiredby the plant. Nuclear encoded enzymes are required in this biosyntheticpathway and are transported from the cytoplasm to the interior of thechloroplast. These nuclear encoded enzymes usually possess an N-terminaltransit peptide that interacts with the chloroplast membrane tofacilitate transport of the peptide to the stroma of the chloroplast.Bruce B. (2000) Chloroplast transit peptides: structure, function, andevolution. Trends Cell Bio. 10:440-447. Upon import, stromal peptidasescleave the transit peptide, leaving the mature functional proteinimported within the chloroplast. Richter S, Lamppa G K. (1999) Stromalprocessing peptidase binds transit peptides and initiates theirATP-dependent turnover in chloroplasts. Journ. Cell Bio. 147:33-43. Thechloroplast transit peptides are variable sequences which are highlydivergent in length, composition and organization. Bruce B. (2000)Chloroplast transit peptides: structure, function, and evolution. TrendsCell Bio. 10:440-447. The sequence similarities of chloroplast transitpeptides diverge significantly amongst homologous proteins fromdifferent plant species. The amount of divergence between chloroplasttransit peptides is unexpected given that the homologous proteinsobtained from different plant species typically share relatively highlevels of sequence similarity when comparing the processed maturefunctional protein.

Novel chimeric chloroplast transit peptide sequences were designed,produced and tested in planta. The novel chimeric chloroplast transitpeptides were shown to possess efficacious translocation and processingproperties for the import of agronomic important proteins within thechloroplast. Initially, native 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS) protein sequences from different plant species wereanalyzed via the ChloroP™ computer program to identify putativechloroplast transit peptide sequences (Emanuelsson O, Nielsen H, vonHeijne G, (1999) ChloroP, a neural network-based method for predictingchloroplast transit peptides and their cleavage sites, Protein Science8; 978-984), available at http://www.cbs.dtu.dk/services/ChloroP/. Afterthe native chloroplast transit peptides were identified, a firstchloroplast transit peptide sequence was aligned with a secondchloroplast transit peptide sequences from a second organism. FIG. 18illustrates the alignment of the EPSPS chloroplast transit peptidesequences of Brassica napus (NCBI Accession No: P17688) and Brassicarapa (NCBI Accession No: AAS80163). Utilizing the chloroplast transitpeptide sequence alignment, novel chimeric chloroplast transit peptideswere designed by combining the first half of the chloroplast transitpeptide sequence from the first organism with the second half of thechloroplast transit peptide sequence from the second organism in anapproximate ratio of 1:1. Exemplary sequences of the newly designedchimeric chloroplast transit peptides are TraP8 (SEQ ID NO:3) and TraP9(SEQ ID NO:4). These novel chimeric chloroplast transit peptidesequences are derived from the EPSPS proteins of Brassica napus [ATCCAccession No: P17688] and Brassica rapa [ATCC Accession No: AAS80163].The TraP8 (SEQ ID NO:3) chimeric chloroplast transit peptide sequencecomprises an N-terminus which is derived from Brassica napus, and theC-terminus of the chloroplast transit peptide is derived from Brassicarapa. The TraP9 (SEQ ID NO:4) chloroplast transit peptide sequencecomprises an N-terminus which is derived from Brassica rapa, and theC-terminus of the chloroplast transit peptide is derived from Brassicanapus. The chimeric chloroplast transit peptides were tested viamultiple assays which included a transient in planta expression systemand transgenically as a stable transformation event comprising a geneexpression element fused to an agronomic important transgene sequence.

Example 2: Transient in Planta Testing of Chimeric Chloroplast TransitPeptide (TraP) Sequences Tobacco Transient Assay:

The Trap8 and TraP9 chimeric chloroplast transit peptide sequences wereinitially tested via a transient in planta assay. Polynucleotidesequences which encode the Trap8 (SEQ ID NO:5) and TraP9 (SEQ ID NO:6)chimeric chloroplast transit peptide sequences were synthesized. Alinker sequence (SEQ ID NO:7) was incorporated between the TraP sequenceand the yfp coding sequence. The resulting constructs contained twoplant transcription units (PTU). The first PTU was comprised of theArabidopsis thaliana Ubiquitin 10 promoter (AtUbi10 promoter; Callis, etal., (1990) J. Biol. Chem., 265: 12486-12493), Trap yellow fluorescentprotein fusion gene (TraP-YFP; US Patent App. 2007/0298412), andAgrobacterium tumefaciens ORF 23 3′ untranslated region (AtuORF23 3′UTR;U.S. Pat. No. 5,428,147). The second PTU was comprised of the CassavaVein Mosaic Virus promoter (CsVMV promoter; Verdaguer et al., (1996)Plant Molecular Biology, 31:1129-1139), phosphinothricin acetyltransferase (PAT; Wohlleben et al., (1988) Gene, 70: 25-37), andAgrobacterium tumefaciens ORF 1 3′ untranslated region (AtuORF1 3′UTR;Huang et al., (1990) J Bacteriol., 172:1814-1822). Construct pDAB101977contains the TraP8 chimeric chloroplast transit peptide (FIG. 2).Construct pDAB101978 contains the TraP9 chimeric chloroplast transitpeptide (FIG. 3). A control plasmid, 101908, which did not contain achloroplast transit peptide sequence upstream of the yfp gene was builtand included in the studies (FIG. 4). The constructs were confirmed viarestriction enzyme digestion and sequencing. Finally, the constructswere transformed into Agrobacterium tumefaciens and stored as glycerolstocks.

From an Agrobacterium glycerol stock, a loop full of frozen culture wasinoculated into 2 ml of YPD (100 μg/ml spectinomycin) in a 14 ml steriletube. The inoculated media was incubated at 28° C. overnight withshaking at 200 rpm. The following day about 100 μl of the culture wasused to inoculate 25 ml of YPD (100 μg/ml spectinomycin) in a 125 mlsterile tri-baffled flask, and incubated overnight at 28° C. overnightwith shaking at 200 rpm. The following day the cultures were diluted toan OD₆₀₀ of 0.5 in sterile ddH₂0 (pH 8.0). The diluted Agrobacteriumstrain was mixed with a second Agrobacterium strain containing the P19helper protein at a ratio of 1:1. The culture were used for tobacco leafinfiltration via the method of Voinnet O, Rivas S, Mestre P, andBaulcombe D., (2003) An enhanced transient expression system in plantsbased on suppression of gene silencing by the p19 protein of tomatobushy stunt virus, The Plant Journal, 33:949-956. Infiltrated tobaccoplants were placed in a Conviron™ set at 16 hr of light at 24° C. for atleast three days until being assayed.

Microscopy Results:

Agrobacterium-infiltrated tobacco leaves were severed from the plant,and placed into a petri-dish with water to prevent dehydration. Theinfiltrated tobacco leaves were observed under blue light excitationwith long-pass filter glasses held in place using a Dark Reader HandLamp™ (Clare Chemical Research Co.; Dolores, Colo.) to identifyundamaged areas of the leaf that were successfully expressing the YFPreporter proteins. Specifically identified leaf areas were dissectedfrom the leaf and mounted in water for imaging by confocal microscopy(Leica TCS-SP5 AOBS™; Buffalo Grove, Ill.). The YFP reporter protein wasexcited by a 514 nm laser line, using a multi-line argon-ion laser. Thewidth of the detection slits was adjusted using a non-expressing (dark)control leaf sample to exclude background leaf autofluoresence.Chlorophyll autofluorescence was simultaneously collected in a secondchannel for direct comparison to the fluorescent reporter protein signalfor determination of chloroplastic localization.

The microscopy imaging results indicated that the YFP fluorescentprotein comprising a TraP8 or TraP9 chloroplast transit peptideaccumulated within the chloroplasts located in the cytoplasm of thetobacco cells as compared to the control YFP fluorescent proteins whichdid not translocate into the chloroplasts of the cytoplasm of thetobacco cells (FIG. 5 and FIG. 6). These microscopy imaging resultssuggest that the translocation of the YFP protein into the chloroplastwas a result of the TraP8 or TraP9 chloroplast transit peptide. As shownin FIG. 5 and FIG. 6 the YFP fluorescence signal is localized in thechloroplasts which also fluoresce red due to auto-fluorescence under themicroscopy imaging conditions. Comparatively, FIG. 7 provides amicroscopy image of tobacco leaf tissue infiltrated with the controlconstruct pDAB101908 that does not contain a chloroplast transitpeptide. The chloroplasts in this image only fluoresce red due toauto-fluorescence under the microscopy imaging conditions, and aredevoid of any YFP fluorescence signal that is exhibited in the TraPinfiltrated tobacco cells. Rather, the YFP fluorescence signal in thecontrol tobacco plant cells is expressed diffusely throughout thecytoplasm of the tobacco plant cells.

Western Blot Results:

Samples of the infiltrated tobacco plants were assayed via Westernblotting. Leaf punches were collected and subjected to bead-milling.About 100-200 mg of leaf material was mixed with 2 BBs (steel balls)(Daisy; Rogers, Ark.) and 500 ml of PBST for 3 minutes in a Kleco™ beadmill. The samples were then spun down in a centrifuge at 14,000×g at 4°C. The supernatant was removed and either analyzed directly via Westernblot or immunoprecipitated. The immunoprecipitations were performedusing the Pierce Direct IP Kit™ (Thermo Scientific; Rockford, Ill.)following the manufacturer's protocol. Approximately, 50 μg of anti-YFPwas bound to the resin. The samples were incubated with the resinovernight at 4° C. Next, the samples were washed and eluted thefollowing morning and prepped for analysis by combining equal volumes of2×8M Urea sample buffer and then boiling the samples for 5 minutes. Theboiled samples were run on a 4-12% SDS-Bis Tris gel in MOPS buffer for40 minutes. The gel was then blotted using the Invitrogen iBlot™ (LifeTechnologies; Carlsbad, Calif.) following the manufacturer's protocol.The blotted membrane was blocked for 10 minutes using 5% non-fat drymilk in PBS-Tween solution. The membrane was probed with the primaryantibody (monoclonal anti-GFP in rabbit) used at a 1:1000 dilution inthe 5% non-fat dry milk in PBS-Tween solution for 1 hour. Next, themembrane was rinsed three times for five minutes with PBS-Tween toremove all unbound primary antibody. The membrane was probed with asecondary monoclonal anti-rabbit in goat antibody (Life Technologies)used at a 1:1000 dilution, for 60 minutes. The membrane was washed aspreviously described and developed by adding Themo BCIP/NBT substrate.The colormetric substrate was allowed to develop for 5-10 minutes andthen the blots were rinsed with water before being dried.

The Western blot results indicated that the YFP protein was expressed inthe infiltrated tobacco cells. Both, the pDAB101977 and pDAB101978infiltrated tobacco plant leaf tissues expressed the YFP protein asindicated by the presence of a protein band which reacted to the YFPantibodies and was equivalent in size to the YFP protein band obtainedfrom tobacco plant leaf tissue infiltrated with the YFP controlconstruct. Moreover, these results indicated that the TraP chimericchloroplast transit peptides were processed and cleaved from the YFPprotein. The TraP8-YFP and TraP9-YFP constructs express a pre-processedprotein band that is larger in molecular weight than the control YFPprotein. The presence of bands on the Western blot which are equivalentin size to the control YFP indicate that the TraP8 and TraP9 chloroplasttransit peptide sequences were processed, thereby reducing the size ofthe YFP to a molecular weight size which is equivalent to the YFPcontrol.

Maize Protoplast Transient Assay:

The Trap8 chimeric chloroplast transit peptide-encoding polynucleotidesequence (SEQ ID NO:5) and the linker-encoding polynucleotide sequence(SEQ ID NO:7) were cloned upstream of the yellow fluorescent proteingene and incorporated into construct pDAB106597 (FIG. 8) for testing viathe maize protoplast transient in planta assay. The resulting constructscontained a single plant transcription unit (PTU). The PTU was comprisedof the Zea mays Ubiquitin 1 promoter (ZmUbi1 promoter; Christensen, A.,Sharrock R., and Quail P., (1992) Maize polyubiquitin genes: structure,thermal perturbation of expression and transcript splicing, and promoteractivity following transfer to protoplasts by electroporation, PlantMolecular Biology, 18:675-689), Trap yellow fluorescent protein fusiongene (TraP8-YFP; US Patent App. 2007/0298412), and Zea mays Peroxidase 53′ untranslated region (ZmPer5 3′UTR; U.S. Pat. No. 6,384,207). Theconstructs were confirmed via restriction enzyme digestion andsequencing.

Seed of Zea mays var. B104 were surface sterilized by shaking vigorouslyin 50% Clorox (3% sodium hypochlorite), containing 2-3 drops of Tween20, for about 20 minutes. The seeds were rinsed thoroughly with steriledistilled water. The sterile seed were plated onto ½ MS medium inPhytatrays or similar type boxes, and allowed to grow in the dark (28°C.) for 12 to 20 days. A maize protoplast transient assay was used toobtain and transfect maize protoplasts from leaves of B104-maize. Thismaize protoplast assay is a modification of the system described by Yoo,S.-D., Cho, Y.-H., and Sheen, J., (2007), Arabidopsis MesophyllProtoplasts: A Versitile Cell System for Transient Gene ExpressionAnalysis, Nature Protocols, 2:1565-1572. The solutions were prepared asdescribed by Yoo et. al., (2007), with the exception that the mannitolconcentration used for the following experiments was change to 0.6 M.

Transfection of 100 to 500 μl of protoplasts (1-5×10⁵) was completed byadding the protoplasts to a 2 ml microfuge tube containing about 40 μgof plasmid DNA (pDAB106597), at room temperature. The volume of DNA waspreferably kept to about 10% of the protoplast volume. The protoplastsand DNA were occasionally mixed during a 5 minute incubation period. Anequal volume of PEG solution was slowly added to the protoplasts andDNA, 2 drops at a time with mixing inbetween the addition of the dropsof PEG solution. The tubes were allowed to incubate for about 10 minuteswith occasional gentle mixing. Next, 1 ml of W5+ solution was added andmixed by inverting the tube several times. The tube(s) were centrifugedfor 5 minutes at 75×g at a temperature of 4° C. Finally, the supernatantwas removed and the pellet was resuspended in 1 ml of WI solution andthe protoplasts were placed into a small Petri plate (35×10 mm) or into6-well multiwell plates and incubated overnight in the dark at roomtemperature. Fluorescence of YFP was viewed by microscopy after 12 hoursof incubation. The microscopy conditions previously described were usedfor the imaging.

The microscopy imaging results indicated that the YFP fluorescentprotein comprising a TraP8 chimeric chloroplast transit peptideaccumulated within the chloroplasts located in the cytoplasm of themaize cells as compared to the control YFP fluorescent proteins whichdid not translocate into the chloroplasts of the cytoplasm of the maizecells (FIG. 9). These microscopy imaging results suggest that thetranslocation of the YFP protein into the chloroplast was a result ofthe TraP8 chimeric chloroplast transit peptide.

Example 3: Chimeric Chloroplast Transit Peptide (TraP) Sequences forExpression of Agronomically Important Transgenes in Arabidopsis

A single amino acid mutation (G96A) in the Escherichia coli5-enolpyruvylshikimate 3-phosphate synthase enzyme (EPSP synthase) canresult in glyphosate insensitivity (Padgette et al., (1991); Eschenburget al., (2002); Priestman et al., (2005); Haghani et al., (2008)). Whilethis mutation confers tolerance to glyphosate, it is also known toadversely affect binding of EPSP synthase with its natural substrate,phosphoenolpyruvate (PEP). The resulting change in substrate bindingefficiency can render a mutated enzyme unsuitable for providing inplanta tolerance to glyphosate.

The NCBI Genbank database was screened in silico for EPSP synthaseprotein and polynucleotide sequences that naturally contain an alanineat an analogous position within the EPSP synthase enzyme as that of theG96A mutation which was introduced into the E. coli version of theenzyme (Padgette et al., (1991); Eschenburg et al., (2002); Priestman etal., (2005); Haghani et al., (2008)).

One enzyme that was identified to contain a natural alanine at thisposition was DGT-28 (GENBANK ACC NO: ZP_06917240.1) from Streptomycessviceus ATCC29083. Further in silico data mining revealed three otherunique Streptomyces enzymes with greater homology to DGT-28; DGT-31(GENBANK ACC NO: YP_004922608.1); DGT-32 (GENBANK ACC NO: ZP_04696613);and DGT-33 (GENBANK ACC NO: NC 010572). Each of these enzymes contains anatural alanine at an analogous position within the EPSP synthase enzymeas that of the G96A mutation that was introduced into the E. coliversion of the enzyme. FIG. 1.

Because EPSP synthase proteins from different organisms are of differentlengths, the numbering of the mutation for the E. coli version of theEPSP synthase enzyme does not necessarily correspond with the numberingof the mutation for the EPSP synthase enzymes from the other organisms.These identified EPSP synthase enzymes were not previously characterizedin regard to glyphosate tolerance or PEP substrate affinity.Furthermore, these EPSP synthase enzymes represent a new class of EPSPsynthase enzymes and do not contain any sequence motifs that have beenused to characterize previously described Class I (plant derivedsequences further described in U.S. Pat. No. RE39247), II (bacteriallyderived sequences further described in U.S. Pat. No. RE39247), and III(bacterially derived sequences further described in International PatentApplication WO 2006/110586) EPSP synthase enzymes.

The novel DGT-14, DGT-28, DGT-31, DGT-32, and DGT-33 enzymes werecharacterized for glyphosate tolerance and PEP substrate affinity bycomparison to Class I EPSP synthase enzymes. The following Class Ienzymes; DGT-1 from Glycine max, DGT-3 from Brassica napus (GENBANK ACCNO: P17688), and DGT-7 from Triticum aestivum (GENBANK ACC NO: EU977181)were for comparison. The Class I EPSP synthase enzymes and mutantvariants thereof were synthesized and evaluated. A mutation introducedinto the plant EPSP synthase enzymes consisted of the Glycine to Alaninemutation made within the EPSP synthase enzyme at a similar location asthat of the G96A mutation from the E. coli version of the enzyme. Inaddition, Threonine to Isoleucine and Proline to Serine mutations wereintroduced within these Class I EPSP synthase enzymes at analogouspositions as that of amino acid 97 (T to I) and amino acid 101 (P to S)in the EPSP synthase of E. coli as described in Funke et al., (2009).

DGT14:

Transgenic T₁ Arabidopsis plants containing the TraP8 and TraP9 chimericchloroplast transit peptides fused to the dgt-14 transgene were producedusing the floral dip method from Clough and Bent (1998), Plant J.16:735-743. Transgenic Arabidopsis plants were obtained and confirmed tocontain the transgene via molecular confirmation. The transgenic plantswere sprayed with differing rates of glyphosate. A distribution ofvarying concentrations of glyphosate rates, including elevated rates,were applied in this study to determine the relative levels ofresistance (105, 420, 1,680 or 3,360 g ae/ha). The typical 1× fieldusage rate of glyphosate is 1,120 g ae/ha. The T₁ Arabidopsis plantsthat were used in this study were variable in copy number for the dgt-14transgene. The low copy dgt-14 T₁ Arabidopsis plants were identifiedusing molecular confirmation assays, and self-pollinated and used toproduce T₂ plants. Table 1 shows the resistance for dgt-14 transgenicplants, as compared to control plants comprising a glyphosate herbicideresistance gene, dgt-1 (as described in U.S. Patent Filing No. 12558351,incorporated herein by reference in its entirety), and wildtypecontrols.

The Arabidopsis T₁ transformants were first selected from the backgroundof untransformed seed using a glufosinate selection scheme. Three flats,or 30,000 seed, were analyzed for each T₁ construct. The selected T₁plants were molecularly characterized and the plants were subsequentlytransplanted to individual pots and sprayed with various rates ofcommercial glyphosate as previously described. The dose response ofthese plants is presented in terms of % visual injury 2 weeks aftertreatment (WAT). Data are presented in the tables below which showindividual plants exhibiting little or no injury (<20%), moderate injury(20-40%), or severe injury (>40%). An arithmetic mean and standarddeviation is presented for each construct used for Arabidopsistransformation. The range in individual response is also indicated inthe last column for each rate and transformation. Wildtype,non-transformed Arabidopsis (c.v. Columbia) served as a glyphosatesensitive control.

The level of plant response varied in the T₁ Arabidopsis plants. Thisvariance can be attributed to the fact each plant represents anindependent transformation event and thus the copy number of the gene ofinterest varies from plant to plant. An overall population injuryaverage by rate is presented in Table 1 to demonstrate the toleranceprovided by each of the dgt-14 constructs linked with either the TraP8v2 or TraP9 v2 chloroplast transit peptide versus the dgt-1 andnon-transformed wildtype controls for varying rates of glyphosate. Theevents contained dgt-14 linked with TraP8 v2 (SEQ ID NO:8) which iscontained in construct pDAB105526 (FIG. 10) and TraP9 v2 (SEQ ID NO:9)which is contained in construct pDAB105527 (FIG. 11). Data from theglyphosate selection of T₁ plants demonstrated that when dgt-14 waslinked with these chloroplast transit peptides, robust tolerance to highlevels of glyphosate was provided. Comparatively, the non-transformed(or wild-type) controls did not provide tolerance to the treatment ofhigh concentrations of glyphosate when treated with similar rates ofglyphosate. In addition, there were instances when events that wereshown to contain three or more copies of dgt-14 were more susceptible toelevated rates of glyphosate. These instances are demonstrated withinthe percent visual injury range shown in Table 1. It is likely that thepresence of high copy numbers of the transgenes within the Arabidopsisplants result in transgene silencing or other epigenetic effects whichresulted in sensitivity to glyphosate, despite the presence of thedgt-14 transgene.

TABLE 1 dgt-14 transformed T₁ Arabidopsis response to a range ofglyphosate rates applied postemergence, compared to a dgt-1 (T₂)segregating population, and a non-transformed control. Visual % injury 2weeks after application. % Injury Range (No. Replicates) % InjuryAnalysis <20% 20-40% >40% Ave Std dev Range (%)  0 20-50  0-70 25-85 0-90  0 0-50 0-70 15-85  5-85 dgt-1 (pDAB3759)  0 30-60   30 40-10045-65  Non-transformed control  0 100 100 100 100

Selected T₁ Arabidopsis plants which were identified to contain low-copynumbers of transgene insertions (1-3 copies) were self-fertilized toproduce a second generation for additional assessment of glyphosatetolerance. The second generation Arabidopsis plants (T₂) which contained1-3 copies of the dgt-14 transgene fused to the TraP8 and TraP9 chimericchloroplast transit peptides were further characterized for glyphosatetolerance and glufosinate tolerance (glufosinate resistance indicatedthat the PAT expression cassette was intact and did not undergorearrangements during the selfing of the T₁ plants). In the T₂generation hemizygous and homozygous plants were available for testingfor each event and therefore were included for each rate of glyphosatetested. Hemizygous plants contain two different alleles at a locus ascompared to homozygous plants which contain the same two alleles at alocus. The copy number and ploidy levels of the T₂ plants were confirmedusing molecular analysis protocols. Likewise, glyphosate was appliedusing the methods and rates as previously described. The dose responseof the plants is presented in terms of % visual injury 2 weeks aftertreatment (WAT). Data are presented as a histogram of individualsexhibiting little or no injury (<20%), moderate injury (20-40%), orsevere injury (>40%). An arithmetic mean and standard deviation arepresented for each construct used for Arabidopsis transformation. Therange in individual response is also indicated in the last column foreach rate and transformation. Wildtype, non-transformed Arabidopsis (cv.Columbia) served as a glyphosate sensitive control. In addition, plantscomprising a glyphosate herbicide resistance gene, dgt-1 (as describedin U.S. Patent Filing No. 12558351, incorporated herein by reference inits entirety) were included as a positive control.

In the T₂ generation both single copy and low-copy (two or three copy)dgt-14 events were characterized for glyphosate tolerance. An overallpopulation injury average by rate is presented in Table 2 to demonstratethe tolerance provided by each of the dgt-14 constructs linked with achloroplast transit peptide versus the dgt-1 and non-transformedwildtype controls for varying rates of glyphosate. The T₂ generationevents contained dgt-14 linked with TraP8 v2 (pDAB105526) and TraP9 v2(pDAB105527). Both of these events are highly resistant to glyphosate.The results indicated that the injury range for the T₂ Arabidopsisplants was less than 20% for all concentrations of glyphosate that weretested. Comparatively, the non-transformed (or wild-type) controls didnot provide tolerance to the treatment of high concentrations ofglyphosate when treated with similar rates of glyphosate. Overall, theresults showed that plants containing and expressing DGT-14 fused to theTraP8 and TraP9 chimeric transit peptide proteins yielded commerciallevel resistance to glyphosate at levels of up to 3 times the field rate(1120 g ae/ha).

TABLE 2 dgt-14 transformed T₂ Arabidopsis response to a range ofglyphosate rates applied postemergence, compared to a dgt-1 (T₂)segregating population, and a non-transformed control. Visual % injury 2weeks after application. Data represents a selected single copy linefrom each construct that segregated as a single locus in theheritability screen. % Injury Range (No. Replicates) % Injury Analysis<20% 20-40% >40% Ave Std dev Range (%) dgt-1 (pDAB3759) Non-transformedcontrol

Randomly selected T₂ Arabidopsis plants which were identified to containlow-copy numbers of transgene insertions (1-3 copies) wereself-fertilized to produce a third generation for additional assessmentof glyphosate tolerance. Arabidopsis seed from the third generation (T₃)were planted and evaluated for glyphosate tolerance using the sameprotocols as previously described. The events tested in the T₃generation contained replicates from each line that were homozygous (asdetermined by using a glufosinate resistance screen to identify if anyof the advanced plants showed segregation of the transgenes). TheseEvents were assayed via LC-MS-MS to confirm that the plants expressedthe DGT-14 protein. The results of the T₃ generation for overallpopulation injury average by rate of glyphosate is presented in Table 3which shows the tolerance to glyphosate provided by each of the dgt-14constructs for varying rates of glyphosate. Exemplary resistant T₃Events comprised dgt-14 linked with TraP8 v2 (pDAB105526) and TraP9 v2(pDAB105527). Both of these Events are highly resistant to glyphosate.The results indicated that the injury range for the T₃ Arabidopsisplants was less than 20% for all concentrations of glyphosate that weretested. Comparatively, the non-transformed (or wild-type) controls didnot provide tolerance to the treatment of high concentrations ofglyphosate when treated with similar rates of glyphosate. Overall, theresults showed that plants containing and expressing DGT-14 yieldedcommercial level resistance to glyphosate at levels of up to 3 times thefield rate (1120 g ae/ha).

TABLE 3 dgt-14 transformed T₃ Arabidopsis response to a range ofglyphosate rates applied postemergence, compared to a dgt-1 (T₂)segregating population, and a non-transformed control. Visual % injury 2weeks after application. Data represents a selected single copypopulation from each construct that segregated as a single locus in theT₂ heritability screen. % Injury Range (No. Replicates) % InjuryAnalysis <20% 20-40% >40% Ave Std dev Range (%) dgt-1 (pDAB3759)Non-transformed control

The data show that expression of a glyphosate-resistant enzyme (e.g.,DGT-28), when targeted to the chloroplast of a plant cell by a TraPtransit peptide in a fusion protein, is capable of conferring glyphosateresistance to the plant cell and plants comprised of these cells.

DGT-28, DGT-31, DGT-32, and DGT-33:

The newly-designed, dicotyledonous plant optimized dgt-28 v5polynucleotide sequence is listed in SEQ ID NO:16. The newly-designed,monocotyledonous plant optimized dgt-28 v6 polynucleotide sequence islisted in SEQ ID NO:17; this sequence was slightly modified by includingan alanine at the second amino acid position to introduce a restrictionenzyme site. The resulting DNA sequences have a higher degree of codondiversity, a desirable base composition, contains strategically placedrestriction enzyme recognition sites, and lacks sequences that mightinterfere with transcription of the gene, or translation of the productmRNA.

Synthesis of DNA fragments comprising SEQ ID NO:16 and SEQ ID NO:17containing additional sequences, such as 6-frame stops (stop codonslocated in all six reading frames that are added to the 3′ end of thecoding sequence), and a 5′ restriction site for cloning were performedby commercial suppliers (DNA2.0, Menlo Park, Calif.). The syntheticnucleic acid molecule was then cloned into expression vectors andtransformed into plants or bacteria as described in the Examples below.

Similar codon optimization strategies were used to design dgt-1, dgt-3v2 (G173A), dgt-3 v3 (G173A; P178S), dgt-3 v4 (T174I; P178S), dgt-7 v4(T168I; P172S), dgt-32 v3, dgt-33 v3, and dgt-31 v3. The codon optimizedversion of these genes are listed as SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQID NO:25, respectively.

Plant Binary Vector Construction.

Standard cloning methods were used in the construction of entry vectorscontaining a chloroplast transit peptide polynucleotide sequence joinedto dgt-28 as an in-frame fusion. The entry vectors containing a transitpeptide (TraP) fused to dgt-28 were assembled using the IN-FUSION™Advantage Technology (Clontech, Mountain View, Calif.). As a result ofthe fusion, the first amino acid, methionine, was removed from dgt-28.Transit peptides TraP4 v2 (SEQ ID NO:26), TraP5 v2 (SEQ ID NO:27), TraP8v2 (SEQ ID NO:28), TraP9 v2 (SEQ ID NO:29), TraP12 v2 (SEQ ID NO:30),and TraP13 v2 (SEQ ID NO:31) were each synthesized by DNA2.0 (MenloPark, Calif.) and fused to the 5′ end fragment of dgt-28, up to andincluding a unique AccI restriction endonuclease recognition site.

Binary plasmids which contained the various TraP and dgt-28 expressioncassettes were driven by the Arabidopsis thaliana Ubiquitin 10 promoter(AtUbi10 v2; Callis, et al., (1990) 1 Biol. Chem., 265: 12486-12493) andflanked by the Agrobacterium tumefaciens open reading frame twenty-three3′ untranslated region (AtuORF23 3′ UTR v1; U.S. Pat. No. 5,428,147).

The assembled TraP and dgt-28 expression cassettes were engineered usingGATEWAY® Technology (Invitrogen, Carlsbad, Calif.) and transformed intoplants via Agrobacterium-mediated plant transformation. Restrictionendonucleases were obtained from New England BioLabs (NEB; Ipswich,Mass.) and T4 DNA Ligase (Invitrogen) was used for DNA ligation. Gatewayreactions were performed using GATEWAY® LR CLONASE® enzyme mix(Invitrogen) for assembling one entry vector into a single destinationvector which contained the selectable marker cassette Cassava VeinMosaic Virus promoter (CsVMV v2; Verdaguer et al., (1996) Plant Mol.Biol., 31: 1129-1139)—DSM-2 (U.S. Pat. App. No.2007/086813)—Agrobacterium tumefaciens open reading frame one 3′untranslated region (AtuORF1 3′ UTR v6; Huang et al., (1990) J.Bacteriol. 172:1814-1822). Plasmid preparations were performed usingNUCLEOSPIN® Plasmid Kit (Macherey-Nagel Inc., Bethlehem, Pa.) or thePlasmid Midi Kit (Qiagen) following the instructions of the suppliers.DNA fragments were isolated using QIAquick™ Gel Extraction Kit (Qiagen)after agarose Tris-acetate gel electrophoresis.

Colonies of all assembled plasmids were initially screened byrestriction digestion of miniprep DNA. Plasmid DNA of selected cloneswas sequenced by a commercial sequencing vendor (Eurofins™ MWG Operon,Huntsville, Ala.). Sequence data were assembled and analyzed using theSEQUENCHER™ software (Gene Codes Corp., Ann Arbor, Mich.).

The following binary constructs express the various TraP:dgt-28 fusiongene sequences: pDAB107527 (FIG. 19) contains TraP4 v2:dgt-28 v5 (SEQ IDNO:32); pDAB105530 (FIG. 20) contains TraP5 v2: dgt-28 v5 (SEQ IDNO:33); pDAB105531 (FIG. 21) contains TraP8 v2: dgt-28 v5 (SEQ IDNO:34); PDAB105532 (FIG. 22) contains TraP9 v2: dgt-28 v5 (SEQ IDNO:35); pDAB105533 (FIG. 23) contains TraP12 v2: dgt-28 v5 (SEQ IDNO:36); and pDAB105534 (FIG. 24) contains TraP13 v2:dgt-28 v5 (SEQ IDNO:37). The dgt-28 v5 sequence of pDAB105534 was modified wherein thefirst codon (GCA) was changed to (GCT).

Additional Plant Binary Vector Construction.

Cloning strategies similar to those described above were used toconstruct binary plasmids which contain dgt-31, dgt-32, dgt-33, dgt-1,dgt-3, and dgt-7.

The microbially derived genes; dgt-31, dgt-32, and dgt-33, were fusedwith different chloroplast transit peptides than previously described.The following chloroplast transit peptides were used; TraP14 v2 (SEQ IDNO:38), TraP23 v2 (SEQ ID NO:39), TraP24 v2 (SEQ ID NO:40). pDAB107532(FIG. 25) contains dgt-32 v3 fused to TraP14 v2 (SEQ ID NO:41),pDAB107534 (FIG. 26) contains dgt-33 v3 fused to TraP24 v2 (SEQ IDNO:42), and pDAB107533 (FIG. 27) contains dgt-31 v3 fused to TraP23 v2(SEQ ID NO:43). The dgt expression cassettes were driven by theArabidopsis thaliana Ubiquitin 10 promoter (AtUbi10 promoter v2) andflanked by the Agrobacterium tumefaciens open reading frame twenty-three3′ untranslated region (AtuORF23 3′ UTR v1). A DSM-2 selectable markercassette containing Cassava Vein Mosaic Virus promoter (CsVMVv2)—DSM-2—Agrobacterium tumefaciens open reading frame one 3′untranslated region (AtuORF1 3′ UTR v6) was also present in the binaryvector.

Additional binaries are constructed wherein dgt-31 v3, dgt-32 v3, anddgt-33 v3 are fused to the previously described chloroplast transitpeptide sequences. For example, the TraP8 v2 sequence is fused to dgt-31v3, dgt-32 v3, and dgt-33 v3, and cloned into binary vectors asdescribed above.

Binary vectors containing the Class I genes (dgt-1, dgt-3, and dgt-7)were constructed. The following binary vectors were constructed andtransformed into plants: pDAB4104 (FIG. 28), which contains the dgt-1 v4sequence as described in U.S. Patent Application Publication No.2011/0124503, which is flanked by the Nicotiana tabacum Osmotinsequences as described in U.S. Patent Application Publication No.2009/0064376; pDAB102715 (FIG. 29); pDAB102716 (FIG. 30); pDAB102717(FIG. 31); and pDAB102785 (FIG. 32). The various TraP chloroplasttransit peptides that were fused to dgt-28, dgt-31, dgt-32, and dgt-33were not added to the Class I genes, as these plant derived sequencespossess native plant chloroplast transit peptides. These vectors aredescribed in further detail in Table 4.

TABLE 4 Description of the binary vectors which contain a Class I EPSPsynthase gene (i.e., dgt-1, dgt-3, or dgt-7). EPSPS Name Descriptionmutation pDAB4104 RB7 MAR v2 :: CsVMV promoter TI PS v2/NtOsm 5′ UTRv2/dgt-1 v4/NtOsm 3′ UTR v2/AtuORF24 3′ UTR v2 :: AtUbi10 promoterv4/pat v3/AtuORF1 3′UTR v3 binary vector pDAB102715 AtUbi10 promoterv2/dgt-3 GA v2/AtuORF23 3′UTR v1 :: CsVMV promoter v2/pat v9/AtuORF13′UTR v6 binary vector pDAB102716 AtUbi10 promoter v2/dgt-3 GA PSv3/AtuORF23 3′UTR v1 :: CsVMV promoter v2/pat v9/AtuORF1 3′UTR v6 binaryvector pDAB102717 AtUbi10 promoter v2/dgt-3 TI PS v4/AtuORF23 3′UTR v1:: CsVMV promoter v2/pat v9/AtuORF1 3′UTR v6 binary vector pDAB102785AtUbi10 promoter v2/dgt-7 TI PS v4/AtuORF23 3′UTR :: CsVMV promoterv2/DSM-2 v2/AtuORF1 3′UTR v6 binary vector

Arabidopsis thaliana Transformation.

Arabidopsis was transformed using the floral dip method from Clough andBent (1998). A selected Agrobacterium colony containing one of thebinary plasmids described above was used to inoculate one or more 100 mLpre-cultures of YEP broth containing spectinomycin (100 mg/L) andkanamycin (50 mg/L). The culture was incubated overnight at 28° C. withconstant agitation at 225 rpm. The cells were pelleted at approximately5000×g for 10 minutes at room temperature, and the resulting supernatantdiscarded. The cell pellet was gently resuspended in 400 mL dunkingmedia containing: 5% (w/v) sucrose, 10 μg/L 6-benzylaminopurine, and0.04% Silwet™ L-77. Plants approximately 1 month old were dipped intothe media for 5-10 minutes with gentle agitation. The plants were laiddown on their sides and covered with transparent or opaque plastic bagsfor 2-3 hours, and then placed upright. The plants were grown at 22° C.,with a 16-hour light/8-hour dark photoperiod. Approximately 4 weeksafter dipping, the seeds were harvested.

Selection of Transformed Plants.

Freshly harvested T₁ seed [containing the dgt and DSM-2 expressioncassettes] was allowed to dry for 7 days at room temperature. T₁ seedwas sown in 26.5×51-cm germination trays, each receiving a 200 mgaliquot of stratified T₁ seed (˜10,000 seed) that had previously beensuspended in 40 mL of 0.1% agarose solution and stored at 4° C. for 2days to complete dormancy requirements and ensure synchronous seedgermination.

Sunshine Mix LP5 was covered with fine vermiculite and subirrigated withHoagland's solution until wet, then allowed to gravity drain. Each 40 mLaliquot of stratified seed was sown evenly onto the vermiculite with apipette and covered with humidity domes for 4-5 days. Domes were removed1 day prior to initial transformant selection using glufosinatepostemergence spray (selecting for the co-transformed DSM-2 gene).

Seven days after planting (DAP) and again 11 DAP, T₁ plants (cotyledonand 2-4-1f stage, respectively) were sprayed with a 0.2% solution ofLiberty herbicide (200 g ai/L glufosinate, Bayer Crop Sciences, KansasCity, Mo.) at a spray volume of 10 mL/tray (703 L/ha) using a DeVilbisscompressed air spray tip to deliver an effective rate of 280 g ai/haglufosinate per application. Survivors (plants actively growing) wereidentified 4-7 days after the final spraying and transplantedindividually into 3-inch pots prepared with potting media (Metro Mix360). Transplanted plants were covered with humidity domes for 3-4 daysand placed in a 22° C. growth chamber as before or moved to directly tothe greenhouse. Domes were subsequently removed and plants reared in thegreenhouse (22±5° C., 50±30% RH, 14 h light:10 dark, minimum 500 μE/m²s¹natural+supplemental light). Molecular confirmation analysis wascompleted on the surviving T₁ plants to confirm that the glyphosatetolerance gene had stably integrated into the genome of the plants.

Molecular Confirmation.

The presence of the dgt-28 and DSM-2 transgenes within the genome ofArabidopsis plants that were transformed with pDAB107527, pDAB105530,pDAB105531, pDAB105532, pDAB105533, or pDAB105534 was confirmed. Thepresence of these polynucleotide sequences was confirmed via hydrolysisprobe assays, gene expression cassette PCR (also described as planttranscription unit PCR PTU PCR), Southern blot analysis, andQuantitative Reverse Transcription PCR analyses.

The T₁ Arabidopsis plants were initially screened via a hydrolysis probeassay, analogous to TAQMAN™, to confirm the presence of the DSM-2 anddgt-28 transgenes. Events were screened via gene expression cassette PCRto determine whether the dgt expression cassette completely integratedinto the plant genomes without rearrangement. The data generated fromthese studies were used to determine the transgene copy number andidentify select Arabidopsis events for self fertilization andadvancement to the T₂ generation. The advanced T₂ Arabidopsis plantswere also screened via hydrolysis probe assays to confirm the presenceand to estimate the copy number of the DSM-2 and dgt genes within theplant chromosome. Finally, a Southern blot assay was used to confirm theestimated copy number on a subset of the T₁ Arabidopsis plants.

Similar assays were used to confirm the presence of the dgt-1 transgenefrom plants transformed with pDAB4101, the presence of the dgt-32transgene from plants transformed with pDAB107532, the presence of thedgt-33 transgene from plants transformed with pDAB107534, the presenceof the dgt-3 transgene from plants transformed with pDAB102715, thepresence of the dgt-3 transgene from plants transformed with pDAB102716,the presence of the dgt-3 transgene from plants transformed withpDAB102717, and the presence of the dgt-7 transgene from plantstransformed with pDAB102785.

Hydrolysis Probe Assay.

Copy number was determined in the T₁ and T₂ Arabidopsis plants using thehydrolysis probe assay described below. Plants with varying numbers oftransgenes were identified and advanced for subsequent glyphosatetolerance studies.

Tissue samples were collected in 96-well plates and lyophilized for 2days. Tissue maceration was performed with a KLECO™ tissue pulverizerand tungsten beads (Environ Metal INC., Sweet Home, Oreg.). Followingtissue maceration, the genomic DNA was isolated in high-throughputformat using the Biosprint™ 96 Plant kit (Qiagen™ Germantown, Md.)according to the manufacturer's suggested protocol. Genomic DNA wasquantified by QUANT-IT™ PICO GREEN DNA ASSAY KIT (Molecular Probes,Invitrogen, Carlsbad, Calif.). Quantified genomic DNA was adjusted toaround 2 ng/μL for the hydrolysis probe assay using a BIOROBOT3000™automated liquid handler (Qiagen, Germantown, Md.). Transgene copynumber determination by hydrolysis probe assay was performed byreal-time PCR using the LIGHTCYCLER® 480 system (Roche Applied Science,Indianapolis, Ind.). Assays were designed for DSM-2, dgt-28 and theinternal reference gene, TAFIII5 (Genbank ID: NC 003075; Duarte et al.,(201) BMC Evol. Biol., 10:61).\

For amplification, LIGHTCYCLER® 480 Probes Master mix (Roche AppliedScience, Indianapolis, Ind.) was prepared at a 1× final concentration ina 10 μL volume multiplex reaction containing 0.1 μM of each primer forDSM-2 and dgt-28, 0.4 μM of each primer for TAFIII5 and 0.2 μM of eachprobe.

Table 5. A two-step amplification reaction was performed with anextension at 60° C. for 40 seconds with fluorescence acquisition. Allsamples were run and the averaged Cycle threshold (Ct) values were usedfor analysis of each sample. Analysis of real time PCR data wasperformed using LightCycler™ software release 1.5 using the relativequant module and is based on the ΔΔCt method. For this, a sample ofgenomic DNA from a single copy calibrator and known 2 copy check wereincluded in each run. The copy number results of the hydrolysis probescreen were determined for the T₁ and T₂ transgenic Arabidopsis plants.

TABLE 5 Primer and probe Information for hydrolysisprobe assay of DSM-2, dgt-28 and internal reference gene (TAFII15).Primer Name Sequence DSM2A 5′ AGCCACATCCCAGTAACGA 3′ (SEQ ID NO: 44)DSM2S 5′ CCTCCCTCTTTGACGCC 3′ (SEQ ID NO: 45) DSM2 Cy5 probe5′ CAGCCCAATGAGGCATCAGC 3′ (SEQ ID NO: 46) DGT28F5′ CTTCAAGGAGATTTGGGATTTGT 3′ (SEQ ID NO: 47) DGT28R5′ GAGGGTCGGCATCGTAT 3′ (SEQ ID NO: 48) UPL154 probe Cat# 04694406001(Roche, Indianapolis, IN) TAFFY-HEX probe5′ AGAGAAGTTTCGACGGATTTCGGGC 3′ (SEQ ID NO: 49) TAFII15-F5′ GAGGATTAGGGTTTCAACGGAG 3′ (SEQ ID NO: 50) TAFII15-R5′ GAGAATTGAGCTGAGACGAGG 3′ (SEQ ID NO: 51)

Dgt-28 Integration Confirmation Via Southern Blot Analysis.

Southern blot analysis was used to establish the integration pattern ofthe inserted T-strand DNA fragment and identify events which containeddgt-28. Data were generated to demonstrate the integration and integrityof the transgene inserts within the Arabidopsis genome. Southern blotdata were used to identify simple integration of an intact copy of theT-strand DNA. Detailed Southern blot analysis was conducted using a PCRamplified probe specific to the dgt-28 gene expression cassette. Thehybridization of the probe with genomic DNA that had been digested withspecific restriction enzymes identified genomic DNA fragments ofspecific molecular weights, the patterns of which were used to identifyfull length, simple insertion T₁ transgenic events for advancement tothe next generation.

Tissue samples were collected in 2 mL conical tubes (Eppendorf™) andlyophilized for 2 days. Tissue maceration was performed with a KLECKO™tissue pulverizer and tungsten beads. Following tissue maceration, thegenomic DNA was isolated using a CTAB isolation procedure. The genomicDNA was further purified using the Qiagen™ Genomic Tips kit. Genomic DNAwas quantified by Quant-IT™ Pico Green DNA assay kit (Molecular Probes,Invitrogen, Carlsbad, Calif.). Quantified genomic DNA was adjusted to 4μg for a consistent concentration.

For each sample, 4 μg of genomic DNA was thoroughly digested with therestriction enzyme SwaI (New England Biolabs, Beverley, Mass.) andincubated at 25° C. overnight, then NsiI was added to the reaction andincubated at 37° C. for 6 hours. The digested DNA was concentrated byprecipitation with Quick Precipitation Solution™ (Edge Biosystems,Gaithersburg, Md.) according to the manufacturer's suggested protocol.The genomic DNA was then resuspended in 25 μL of water at 65° C. for 1hour. Resuspended samples were loaded onto a 0.8% agarose gel preparedin 1×TAE and electrophoresed overnight at 1.1 V/cm in 1×TAE buffer. Thegel was sequentially subjected to denaturation (0.2 M NaOH/0.6 M NaCl)for 30 minutes, and neutralization (0.5 M Tris-HCl (pH 7.5)/1.5 M NaCl)for 30 minutes.

Transfer of DNA fragments to nylon membranes was performed by passivelywicking 20×SSC solution overnight through the gel onto treatedIMMOBILON™ NY+ transfer membrane (Millipore, Billerica, Mass.) by usinga chromatography paper wick and paper towels. Following transfer, themembrane was briefly washed with 2×SSC, cross-linked with theSTRATALINKER™ 1800 (Stratagene, LaJolla, Calif.), and vacuum baked at80° C. for 3 hours.

Blots were incubated with pre-hybridization solution (Perfect Hyb plus,Sigma, St. Louis, Mo.) for 1 hour at 65° C. in glass roller bottlesusing a model 400 hybridization incubator (Robbins Scientific,Sunnyvale, Calif.). Probes were prepared from a PCR fragment containingthe entire coding sequence. The PCR amplicon was purified using QIAEX™II gel extraction kit and labeled with α³²P-dCTP via the Random RT PrimeIT™ labeling kit (Stratagene, La Jolla, Calif.). Blots were hybridizedovernight at 65° C. with denatured probe added directly to hybridizationbuffer to approximately 2 million counts per blot per mL. Followinghybridization, blots were sequentially washed at 65° C. with0.1×SSC/0.1% SDS for 40 minutes. Finally, the blots were exposed tostorage phosphor imaging screens and imaged using a Molecular DynamicsStorm 860™ imaging system.

The Southern blot analyses completed in this study were used todetermine the copy number and confirm that selected events contained thedgt-28 transgene within the genome of Arabidopsis.

dgt-28 Gene Expression Cassette Confirmation Via PCR Analysis.

The presence of the dgt-28 gene expression cassette contained in the T₁plant events was detected by an end point PCR reaction. Primers (Table6) specific to the AtUbi10 promoter v2 and AtuORF23 3′UTR v1 regions ofthe dgt-28 gene expression cassette were used for detection.

TABLE 6 Oligonucleotide primers used for dgt-28 geneexpression cassette confirmation. Primer Name Sequence Forward oligo5′ CTGCAGGTCAACGGATCAGGATAT 3′ (SEQ ID NO: 52) Reverse oligo5′ TGGGCTGAATTGAAGACATGCTCC 3′ (SEQ ID NO: 53)

The PCR reactions required a standard three step PCR cycling protocol toamplify the gene expression cassette. All of the PCR reactions werecompleted using the following PCR conditions: 94° C. for three minutesfollowed by 35 cycles of 94° C. for thirty seconds, 60° C. for thirtyseconds, and 72° C. for three minutes. The reactions were completedusing the EX-TAQ™ PCR kit (TaKaRa Biotechnology Inc. Otsu, Shiga, Japan)per manufacturer's instructions. Following the final cycle, the reactionwas incubated at 72° C. for 10 minutes. TAE agarose gel electrophoresiswas used to determine the PCR amplicon size. PCR amplicons of anexpected size indicated the presence of a full length gene expressioncassette was present in the genome of the transgenic Arabidopsis events.

dgt-28 Relative Transcription Confirmation Via Quantitative ReverseTranscription PCR Analysis.

Tissue samples of dgt-28 transgenic plants were collected in 96-wellplates and frozen at 80° C. Tissue maceration was performed with aKLECO™ tissue pulverizer and tungsten beads (Environ Metal INC., SweetHome, Oreg.). Following tissue maceration, the Total RNA was isolated inhigh-throughput format using the Qiagen™ Rneasy 96 kit (Qiagen™,Germantown, Md.) according to the manufacturer's suggested protocolwhich included the optional DnaseI treatment on the column. This stepwas subsequently followed by an additional DnaseI (Ambion™, Austin,Tex.) treatment of the eluted total RNA. cDNA synthesis was carried outusing the total RNA as template with the High Capacity cDNA ReverseTranscription™ kit (Applied Biosystems, Austin, Tex.) following themanufacturer's suggested procedure with the addition of theoligonucleotide, TVN. Quantification of expression was completed byhydrolysis probe assay and was performed by real-time PCR using theLIGHTCYCLER®480 system (Roche Applied Science, Indianapolis, Ind.).Assays were designed for dgt-28 and the internal reference gene “unknownprotein” (Genbank Accession Number: AT4G24610) using the LIGHTCYCLER®Probe Design Software 2.0. For amplification, LIGHTCYCLER®480 ProbesMaster mix (Roche Applied Science, Indianapolis, Ind.) was prepared at1× final concentration in a 10 μL volume singleplex reaction containing0.4 μM of each primer, and 0.2 μM of each probe. Table 7.

TABLE 7 PCR primers used for quantitative reversetranscription PCR analysis of dgt-28. Primer Name Sequence AT26410LP5′ CGTCCACAAAGCTGAATGTG 3′ (SEQ ID NO: 54) AT26410RP5′ CGAAGTCATGGAAGCCACTT3′ (SEQ ID NO: 55) UPL146 Cat# 04694325001(Roche, Indianapolis, IN) DGT28F 5′ CTTCAAGGAGATTTGGGATTTGT3′(SEQ ID NO: 56) DGT28R 5′ GAGGGTCGGCATCGTAT 3′ (SEQ ID NO: 57)UPL154 probe Cat# 04694406001 (Roche, Indianapolis, IN)

A two-step amplification reaction was performed with an extension at 60°C. for 40 seconds with fluorescence acquisition. All samples were run intriplicate and the averaged Cycle threshold (Ct) values were used foranalysis of each sample. A minus reverse transcription reaction was runfor each sample to ensure that no gDNA contamination was present.Analysis of real time PCR data was performed based on the ΔΔCt method.This assay was used to determine the relative expression of dgt-28 intransgenic Arabidopsis events which were determined to be hemizygous andhomozygous. The relative transcription levels of the dgt-28 mRNA rangedfrom 2.5 fold to 207.5 fold higher than the internal control. These dataindicate that dgt-28 transgenic plants contained a functional dgt-28gene expression cassette, and the plants were capable of transcribingthe dgt-28 transgene.

Western Blotting Analysis.

DGT-28 was detected in leaf samples obtained from transgenic Arabidopsisthaliana plants. Plant extracts from dgt-28 transgenic plants and DGT-28protein standards were incubated with NUPAGE® LDS sample buffer(Invitrogen, Carlsbad, Calif.) containing DTT at 90° C. for 10 minutesand electrophoretically separated in an acrylamide precast gel. Proteinswere then electro-transferred onto nitrocellulose membrane using themanufacturer's protocol. After blocking with the WESTERNBREEZE® BlockingMix (Invitrogen) the DGT-28 protein was detected by anti-DGT-28antiserum followed by goat anti-rabbit phosphatase. The detected proteinwas visualized by chemiluminescence substrate BCIP/NBT Western AnalysisReagent (KPL, Gaithersburg, Md.). Production of an intact DGT-28 proteinvia Western blot indicated that the dgt-28 transgenic plants which wereassayed expressed the DGT-28 protein.

Transgenic T₁ Arabidopsis plants containing the dgt-28 transgene weresprayed with differing rates of glyphosate. Elevated rates were appliedin this study to determine the relative levels of resistance (105, 420,1,680 or 3,360 g ae/ha). A typical 1× usage rate of glyphosate that willcontrol non-transformed Arabidopsis is 420 g ae/ha. Glyphosateformulations with the addition of ammonium sulfate were applied to theT₁ plants with a track sprayer calibrated at 187 L/ha. The T₁Arabidopsis plants that were used in this study were variable copynumber for the dgt-28 transgene. The low copy dgt-28 T₁ Arabidopsisplants were self-pollinated and used to produce T₂ plants. Table 8 showsthe comparison of dgt-28 transgenic plants, drawn to a glyphosateherbicide resistance gene, dgt-1, and wildtype controls. Table 9 showsthe comparison of dgt-32, and dgt-33 drawn to a glyphosate herbicideresistance gene, dgt-1, and wildtype controls. Table 10 shows thecomparison of the novel bacterial EPSP synthase enzymes to the Class IEPSP synthase enzymes and the controls at a glyphosate rate of 1,680 gae/ha.

Results of Glyphosate Selection of Transformed Dgt-28 ArabidopsisPlants.

The Arabidopsis T₁ transformants were first selected from the backgroundof untransformed seed using a glufosinate selection scheme. Three flatsor 30,000 seed were analyzed for each T₁ construct. The T₁ plantsselected above were molecularly characterized and representative plantswith variable copy number were subsequently transplanted to individualpots and sprayed with various rates of commercial glyphosate aspreviously described. The response of these plants is presented in termsof % visual injury 2 weeks after treatment (WAT). Data are presented ina table which shows individual plants exhibiting little or no injury(<20%), moderate injury (20-40%), or severe injury (>40%). An arithmeticmean and standard deviation is presented for each construct used forArabidopsis transformation. The range in individual response is alsoindicated in the last column for each rate and transformation.Wild-type, non-transformed Arabidopsis (c.v. Columbia) served as aglyphosate sensitive control.

The level of plant response varied. This variance can be attributed tothe fact each plant represents an independent transformation event andthus the copy number of the gene of interest varies from plant to plant.It was noted that some plants which contained the transgene were nottolerant to glyphosate; a thorough analysis to determine whether theseplants expressed the transgene was not completed. It is likely that thepresence of high copy numbers of the transgene within the T₁ Arabidopsisplants resulted in transgene silencing or other epigenetic effects whichresulted in sensitivity to glyphosate, despite the presence of thedgt-28 transgene.

An overall population injury average by rate is presented in Table 10for rates of glyphosate at 1,680 g ae/ha to demonstrate the significantdifference between the plants transformed with dgt-3, dgt-7, dgt-28,dgt-32, and dgt-33 versus the dgt-1 and wild-type controls.

The tolerance provided by the novel bacterial EPSP synthases varieddepending upon the specific enzyme. DGT-28, DGT-32, and DGT-33unexpectedly provided significant tolerance to glyphosate. The dgt genesimparted herbicide resistance to individual T₁ Arabidopsis plants acrossall transit peptides tested. As such, the use of additional chloroplasttransit peptides (i.e., TraP8-dgt-32 or TraP8-dgt-33) would provideprotection to glyphosate with similar injury levels as reported within agiven treatment.

TABLE 8 dgt-28 transformed T₁ Arabidopsis response to a range ofglyphosate rates applied postemergence, compared to a dgt-1 (T₄)homozygous resistant population, and a non-transformed control. Visual %injury 14 days after application. % Injury % Injury Averages <20%20-40% >40% Ave Std dev Range (%) pDAB107527: TraP4 v2 - dgt-28 v5 0 gae/ha glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha glyphosate 4 0 0 3.8 7.50-15 420 g ae/ha glyphosate 2 1 1 28.8 28.1 0-65 1680 g ae/ha glyphosate0 2 2 55.0 26.8 35-85  3360 g ae/ha glyphosate 0 2 2 43.8 18.0 30-70 pDAB105530: TraP5 v2 - dgt-28 v5 0 g ae/ha glyphosate 6 0 0 0.0 0.0 0105 g ae/ha glyphosate 2 2 2 39.3 37.4  8-100 420 g ae/ha glyphosate 1 41 33.0 26.6 8-85 1680 g ae/ha glyphosate 0 4 2 47.5 27.5 25-85  3360 gae/ha glyphosate 0 0 6 76.7 13.7 50-85  pDAB105531: TraP8 v2 - dgt-28 v50 g ae/ha glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha glyphosate 3 1 0 10.810.4 0-25 420 g ae/ha glyphosate 3 0 1 22.8 18.6 8-50 1680 g ae/haglyphosate 4 0 0 5.3 3.8 0-8  3360 g ae/ha glyphosate 0 4 0 29.3 6.822-35  pDAB105532: TraP9 v2 - dgt-28 v5 0 g ae/ha glyphosate 4 0 0 0.00.0 0 105 g ae/ha glyphosate 3 0 1 17.5 28.7 0-60 420 g ae/ha glyphosate1 1 2 39.5 25.1 18-70  1680 g ae/ha glyphosate 3 0 1 26.3 36.1 5-80 3360g ae/ha glyphosate 3 0 1 25.8 32.9 8-75 pDAB105533: TraP12 v2 - dgt-28v5 0 g ae/ha glyphosate 5 0 0 0.0 0.0 0 105 g ae/ha glyphosate 4 1 010.0 10.0 0-25 420 g ae/ha glyphosate 1 1 3 53.6 34.6 8-85 1680 g ae/haglyphosate 4 1 0 11.0 8.2 0-20 3360 g ae/ha glyphosate 0 2 3 55.0 25.525-80  pDAB105534: TraP13 v2 - dgt-28 v5 0 g ae/ha glyphosate 5 0 0 0.00.0 0 105 g ae/ha glyphosate 4 0 1 14.0 20.6 0-50 420 g ae/ha glyphosate3 1 1 17.6 19.5 0-50 1680 g ae/ha glyphosate 3 0 2 39.0 47.1  5-100 3360g ae/ha glyphosate 2 2 1 31.2 22.3 18-70  pDAB4104: dgt-1 (transformedcontrol) 0 g ae/ha glyphosate 5 0 0 0.0 0.0 0 105 g ae/ha glyphosate 0 04 80.0 0.0 80  420 g ae/ha glyphosate 0 0 4 80.0 0.0 80  1680 g ae/haglyphosate 0 0 4 80.0 0.0 80  3360 g ae/ha glyphosate 0 0 4 81.3 2.580-85  WT (non-transformed control) 0 g ae/ha glyphosate 5 0 0 0.0 0.0 0105 g ae/ha glyphosate 0 0 4 100.0 0.0 100  420 g ae/ha glyphosate 0 0 4100.0 0.0 100  1680 g ae/ha glyphosate 0 0 4 100.0 0.0 100  3360 g ae/haglyphosate 0 0 4 100.0 0.0 100 

TABLE 9 dgt-32, and dgt-33 transformed T₁ Arabidopsis response to arange of glyphosate rates applied postemergence, compared to a dgt-1(T₄) homozygous resistant population, and a non-transformed control.Visual % injury 14 days after application. % Injury % Injury Averages<20% 20-40% >40% Ave Std dev Range (%) pDAB107532: TraP14 v2 - dgt-32 v30 g ae/ha glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha glyphosate 4 0 0 0.00.0 0 420 g ae/ha glyphosate 2 0 2 30.0 29.4 0-60 1680 g ae/haglyphosate 3 0 1 17.5 21.8 5-50 3360 g ae/ha glyphosate 0 3 1 35.0 30.020-80  pDAB107534: TraP24 v2 - dgt-33 v3 0 g ae/ha glyphosate 4 0 0 0.00.0 0 105 g ae/ha glyphosate 2 2 0 21.3 14.9 5-40 420 g ae/ha glyphosate1 1 2 46.3 30.9 5-70 1680 g ae/ha glyphosate 1 0 3 62.5 38.8 5-90 3360 gae/ha glyphosate 1 0 3 62.0 36.0 8-80 pDAB4104: dgt-1 (transformedcontrol) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha glyphosate 0 23 42.5 15.0 20-50  420 g ae/ha glyphosate 0 1 2 38.8 11.1 25-50  1680 gae/ha glyphosate 0 0 4 79.0 19.4 50-90  3360 g ae/ha glyphosate 0 0 450.0 0.0 50 WT (non-transformed control) 0 g ae/ha glyphosate 4 0 0 0.00.0 0 105 g ae/ha glyphosate 0 0 4 85.0 0.0 85  420 g ae/ha glyphosate 00 4 100.0 0.0 100  1680 g ae/ha glyphosate 0 0 4 100.0 0.0 100  3360 gae/ha glyphosate 0 0 4 100.0 0.0 100 

TABLE 10 dgt-28, dgt-32, dgt-33, dgt-3, and dgt-7 transformed T₁Arabidopsis response to glyphosate applied postemergence at 1,680 gae/ha, compared to a dgt-1 (T₄) homozygous resistant population, and anon-transformed control. Visual % injury 14 days after application. %Injury % Injury <20% 20-40% >40% Ave Std dev Range (%) BacterialpDAB07527 TraP4 v2 - dgt-28 v5 0 2 2 55.0 26.8 35-85  Enzymes pDAB105530TraP5 v2 - dgt -28 v5 0 4 2 47.5 27.5 25-85  pDAB105531 TraP8 v2 - dgt-28 v5 4 0 0 5.3 3.8 0-8  pDAB105532 TraP9 v2 - dgt -28 v5 3 0 1 26.336.1 5-80 pDAB105533 Trap12 v2 - dgt -28 v5 4 1 0 11.0 8.2 0-20pDAB105534 TraP13 v2 - dgt -28 v5 3 0 2 39.0 47.1  5-100 pDAB107532TraP14 v2 - dgt-32 v3 3 0 1 17.5 21.8 5-50 pDAB107534 TraP24 v2 - dgt-33v3 1 0 3 62.5 38.8 5-90 Class I pDAB102715 dgt-3 v2 4 0 3 42 48  0-100Enzymes pDAB102716 dgt-3 v3 2 0 1 14 23 0-40 pDAB102717 dgt-3 v4 3 2 128 35 10-100 pDAB102785 dgt-7 v4 0 1 1 45 21 30-60  pDAB4104 dgt-1(transformed 0 0 4 80.0 0.0  80 control) — WT (non-transfor 0 0 4 100.00.0 100 med control)

dgt-28 as a Selectable Marker.

The use of dgt-28 as a selectable marker for glyphosate selection agentis tested with the Arabidopsis transformed plants described above.Approximately 50 T₄ generation Arabidopsis seed (homozygous for dgt-28)are spiked into approximately 5,000 wildtype (sensitive to glyphosate)seed. The seeds are germinated and plantlets are sprayed with aselecting dose of glyphosate. Several treatments of glyphosate arecompared; each tray of plants receives either one or two applicationtimings of glyphosate in one of the following treatment schemes: 7 DAP(days after planting), 11 DAP, or 7 followed by 11 DAP. Since all plantsalso contain a glufosinate resistance gene in the same transformationvector, dgt-28 containing plants selected with glyphosate can bedirectly compared to DSM-2 or pat containing plants selected withglufosinate.

Glyphosate treatments are applied with a DeVilbiss™ spray tip aspreviously described. Transgenic plants containing dgt-28 are identifiedas “resistant” or “sensitive” 17 DAP. Treatments of 26.25-1680 g ae/haglyphosate applied 7 and 11 days after planting (DAP), show effectiveselection for transgenic Arabidopsis plants that contain dgt-28.Sensitive and resistant plants are counted and the number of glyphosatetolerant plants is found to correlate with the original number oftransgenic seed containing the dgt-28 transgene which are planted. Theseresults indicate that dgt-28 can be effectively used as an alternativeselectable marker for a population of transformed Arabidopsis.

Heritability.

Confirmed transgenic T₁ Arabidopsis events were self-pollinated toproduce T₂ seed. These seed were progeny tested by applying Ignite™herbicide containing glufosinate (200 g ae/ha) to 100 random T₂siblings. Each individual T₂ plant was transplanted to 7.5-cm squarepots prior to spray application (track sprayer at 187 L/ha applicationsrate). The T₁ families (T₂ plants) segregated in the anticipated 3Resistant: 1 Sensitive model for a dominantly inherited single locuswith Mendelian inheritance as determined by Chi square analysis(P>0.05). The percentage of T₁ families that segregated with theexpected Mendelian inheritance are illustrated in Table 11, anddemonstrate that the dgt-28 trait is passed via Mendelian inheritance tothe T₂ generation. Seed were collected from 5 to 15 T₂ individuals (T₃seed). Twenty-five T₃ siblings from each of 3-4 randomly-selected T₂families were progeny tested as previously described. Data showed nosegregation and thus demonstrated that dgt-28 and dgt-3 are stablyintegrated within the chromosome and inherited in a Mendelian fashion toat least three generations.

TABLE 11 Percentage of T₁ families (T₂ plants) segregating as singleMendelian inheritance for a progeny test of 100 plants. T1 FamiliesTested Gene of Interest Segregating at 1 Locus (%) dgt-3 v2 64% dgt-3 v360% dgt-3 v4 80% dgt-7 v4 63% TraP5 v2 - dgt-28 v5 100%  TraP8 v2 -dgt-28 v5 100%  TraP9 v2 - dgt-28 v5 100%  TraP12 v2 - dgt-28 v5 50%TraP13 v2 - dgt-28 v5 75% yfp Transgenic Control 100%  Plants

T₂ Arabidopsis Data.

The second generation plants (T₂) of selected T₁ Arabidopsis eventswhich contained low copy numbers of the dgt-28 transgene were furthercharacterized for glyphosate tolerance. Glyphosate was applied asdescribed previously. The response of the plants is presented in termsof % visual injury 2 weeks after treatment (WAT). Data are presented asa histogram of individuals exhibiting little or no injury (<20%),moderate injury (20-40%), or severe injury (>40%). An arithmetic meanand standard deviation are presented for each construct used forArabidopsis transformation. The range in individual response is alsoindicated in the last column for each rate and transformation.Wild-type, non-transformed Arabidopsis (cv. Columbia) served as aglyphosate sensitive control. In the T₂ generation hemizygous andhomozygous plants were available for testing for each event andtherefore were included for each rate of glyphosate tested. Hemizygousplants contain two different alleles at a locus as compared tohomozygous plants which contain the same two alleles at a locus.Variability of response to glyphosate is expected in the T₂ generationas a result of the difference in gene dosage for hemizygous as comparedto homozygous plants. The variability in response to glyphosate isreflected in the standard deviation and range of response.

In the T₂ generation both single copy and multi-copy dgt-28 events werecharacterized for glyphosate tolerance. Within an event, single copyplants showed similar levels of tolerance to glyphosate. Characteristicdata for a single copy T₂ event are presented in Table 12. Eventscontaining dgt-28 linked with TraP5 v2 did not provide robust toleranceto glyphosate as compared with the dgt-28 constructs which containedother TraP transit peptides. However, the dgt-28 TraP5 constructs didprovide a low level of glyphosate tolerance as compared to thenon-transformed Columbia control. There were instances when events thatwere shown to contain two or more copies of dgt-28 were more susceptibleto elevated rates of glyphosate (data not shown). This increase insensitivity to glyphosate is similar to the data previously describedfor the T₁ plants which also contained high copy numbers of the dgt-28transgene. It is likely that the presence of high copy numbers of thetransgene within the Arabidopsis plants result in transgene silencing orother epigenetic effects which resulted in sensitivity to glyphosate,despite the presence of the dgt-28 transgene.

These events contained dgt-28 linked with TraP5 v2 (pDAB105530), TraP12v2 (pDAB105533) and TraP13 v2 (pDAB105534).

In addition to dgt-28, T₂ Arabidopsis events transformed with dgt-3 arepresented in Table 13. As described for the dgt-28 events in Table 12,the data table contains a representative event that is characteristic ofthe response to glyphosate for each construct. For the dgt-3characterization, constructs containing a single PTU (planttransformation unit) with the dgt-3 gene being driven by the AtUbi10promoter (pDAB102716, FIG. 30 and pDAB102715, FIG. 29) were compared toconstructs with the same gene containing 2 PTUs of the gene (pDAB102719,FIG. 33; pDAB102718, FIG. 34). The constructs which contained 2 PTU usedthe AtUbi10 promoter to drive one copy of the gene and the CsVMVpromoter to drive the other copy. The use of the double PTU wasincorporated to compare the dgt-3 transgenic plants with dgt-28transgenic plants which contained two copies of the transgene. Datademonstrated that single copy T₂ dgt-3 events with only a single PTUwere more susceptible to glyphosate than single copy dgt-28 eventstested, but were more tolerant than the non-transformed control. T₁families containing 2 PTUs of the dgt-3 gene provided a higher level ofvisual tolerance to glyphosate compared to the 1 PTU constructs. In bothinstances the T₁ families were compared to the dgt-1 and wildtypecontrols. T₂ data demonstrate that dgt-28 provides robust tolerance assingle copy events.

TABLE 12 Response of selected individual T₂ Arabidopsis eventscontaining dgt-28 to glyphosate applied postemergence at varying rates,compared to a dgt-1 (T₄) homozygous resistant population, and anon-transformed control. Visual % injury 14 days after application. %Injury % Injury 1 copy <20% 20-40% >40% Ave Std dev Range (%)pDAB105530: TraP5 v2 - dgt-28 v5 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0420 g ae/ha glyphosate 0 0 4 75.0 17.8 50-90 840 g ae/ha glyphosate 0 04 80.0 20.0 50-90 1680 g ae/ha glyphosate 0 0 4 75.0 10.8 60-85 3360 gae/ha glyphosate 0 0 4 76.3 4.8 70-80 pDAB105531: TraP8 v2 - dgt-28 v5 0g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g ae/ha glyphosate 4 0 0 0.5 1.00-2 840 g ae/ha glyphosate 4 0 0 1.3 2.5 0-5 1680 g ae/ha glyphosate 4 00 7.5 5.0  5-15 3360 g ae/ha glyphosate 4 0 0 7.5 6.5  0-15 pDAB105532:TraP9 v2 - dgt-28 v5 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g ae/haglyphosate 4 0 0 2.0 4.0 0-8 840 g ae/ha glyphosate 4 0 0 9.0 2.0  8-121680 g ae/ha glyphosate 4 0 0 7.3 4.6  2-12 3360 g ae/ha glyphosate 4 00 11.0 1.2 10-12 pDAB105533: TraP12 v2 - dgt-28 v5 0 g ae/ha glyphosate4 0 0 0.0 0.0 0 420 g ae/ha glyphosate 4 0 0 0.0 0.0 0 840 g ae/haglyphosate 4 0 0 0.0 0.0 0 1680 g ae/ha glyphosate 4 0 0 0.0 0.0 0 3360g ae/ha glyphosate 3 1 0 13.3 7.9  8-25 pDAB105534: TraP13 v2 - dgt-28v5 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g ae/ha glyphosate 3 1 0 5.010.0  0-20 840 g ae/ha glyphosate 3 1 0 5.0 10.0  0-20 1680 g ae/haglyphosate 2 2 0 10.0 11.5  0-20 3360 g ae/ha glyphosate 2 2 0 15.0 12.2 5-30 WT (non-transformed control) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0420 g ae/ha glyphosate 0 0 4 100.0 0.0 100  840 g ae/ha glyphosate 0 0 4100.0 0.0 100  1680 g ae/ha glyphosate 0 0 4 100.0 0.0 100  3360 g ae/haglyphosate 0 0 4 100.0 0.0 100  pDAB4104: dgt-1 (transformed control) 0g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g ae/ha glyphosate 0 4 0 37.5 2.935-40 840 g ae/ha glyphosate 0 0 4 45.0 0.0 45  1680 g ae/ha glyphosate0 0 4 47.5 2.9 45-50 3360 g ae/ha glyphosate 0 0 4 50.0 0.0 50 

TABLE 13 Response of selected T₂ Arabidopsis events transformed withdgt-3 to glyphosate applied postemergence at varying rates. Visual %injury 14 days after application. % Injury % Injury 1 copy seg <20%20-40% >40% Ave Std dev Range (%) pDAB102716: dgt-3 v3 (1 PTU) 0 g ae/haglyphosate 4 0 0 0 0 0 420 g ae/ha glyphosate 1 1 2 39 25 15-65 840 gae/ha glyphosate 0 2 2 50 23 30-70 1680 g ae/ha glyphosate 0 1 3 69 1940-80 3360 g ae/ha glyphosate 0 0 4 79 6 70-85 pDAB102719: dgt-3 v3 (2PTU) 0 g ae/ha glyphosate 4 0 0 0 0 0 420 g ae/ha glyphosate 0 4 0 20 020 840 g ae/ha glyphosate 0 3 1 38 5 35-45 1680 g ae/ha glyphosate 3 1 015 7 10-25 3360 g ae/ha glyphosate 2 2 0 21 8 15-30 pDAB102715: dgt-3 v2(1 PTU) 0 g ae/ha glyphosate 4 0 0 0 0 0 420 g ae/ha glyphosate 2 2 0 2616 10-40 840 g ae/ha glyphosate 0 2 2 55 17 40-70 1680 g ae/haglyphosate 0 2 2 56 22 35-75 3360 g ae/ha glyphosate 0 0 4 65 17 50-80pDAB102718: dgt-3 v2 (2 PTU) 0 g ae/ha glyphosate 4 0 0 0 0 0 420 gae/ha glyphosate 4 0 0 5 7  0-15 840 g ae/ha glyphosate 2 2 0 23 1015-35 1680 g ae/ha glyphosate 3 0 1 20 20  5-50 3360 g ae/ha glyphosate1 1 2 36 22 15-60

T₃ Arabidopsis Data.

The third generation plants (T₃) of selected T₂ Arabidopsis events whichcontained low copy numbers of the dgt-28 transgene were furthercharacterized for glyphosate tolerance. Twenty-five plants per line wereselected with glufosinate as previously described and lines from everyconstruct tested did not segregate for the selectable marker gene.Glyphosate was applied as described previously. The response of theplants is presented in terms of % visual injury 2 weeks after treatment(WAT). Data are presented as a histogram of individuals exhibitinglittle or no injury (<20%), moderate injury (20-40%), or severe injury(>40%). An arithmetic mean and standard deviation are presented for eachconstruct used for Arabidopsis transformation. The range in individualresponse is also indicated in the last column for each rate andtransformation. Wild-type, non-transformed Arabidopsis (cv. Columbia)served as a glyphosate-sensitive control.

TABLE 14 Response of selected individual T₃ Arabidopsis eventscontaining dgt-28 to glyphosate applied postemergence at varying rates,compared to a dgt-1 (T₄) homozygous resistant population, and anon-transformed control. Visual % injury 14 days after application. %Injury Range (No. Replicates) % Injury Analysis <20% 20-40% >40% Ave Stddev Range (%)

Selection of Transformed Plants.

Freshly harvested T₁ seed [dgt-31, dgt-32, and dgt-33 v1 gene] wereallowed to dry at room temperature and shipped to Indianapolis fortesting. T₁ seed was sown in 26.5×51-cm germination trays (T.O. PlasticsInc., Clearwater, Minn.), each receiving a 200 mg aliquots of stratifiedT₁ seed (˜10,000 seed) that had previously been suspended in 40 mL of0.1% agarose solution and stored at 4° C. for 2 days to completedormancy requirements and ensure synchronous seed germination.

Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, Wash.) wascovered with fine vermiculite and subirrigated with Hoagland's solutionuntil wet, then allowed to gravity drain. Each 40 mL aliquot ofstratified seed was sown evenly onto the vermiculite with a pipette andcovered with humidity domes (KORD™ Products, Bramalea, Ontario, Canada)for 4-5 days. Domes were removed once plants had germinated prior toinitial transformant selection using glufosinate postemergence spray(selecting for the co-transformed dsm-2 gene).

Six days after planting (DAP) and again 10 DAP, T₁ plants (cotyledon and2-4-1f stage, respectively) were sprayed with a 0.1% solution of IGNITE™herbicide (280 g ai/L glufosinate, Bayer Crop Sciences, Kansas City,Mo.) at a spray volume of 10 mL/tray (703 L/ha) using a DeVilbiss™compressed air spray tip to deliver an effective rate of 200 g ae/haglufosinate per application. Survivors (plants actively growing) wereidentified 4-7 days after the final spraying. Surviving plants weretransplanted individually into 3-inch pots prepared with potting media(Metro Mix 360™). Plants reared in the greenhouse at least 1 day priorto tissue sampling for copy number analyses.

T₁ plants were sampled and copy number analysis for the dgt-31, dgt-32,and dgt-33 v1 gene were completed. T₁ plants were then assigned tovarious rates of glyphosate so that a range of copies were among eachrate. For Arabidopsis, 26.25 g ae/ha glyphosate is an effective dose todistinguish sensitive plants from ones with meaningful levels ofresistance. Elevated rates were applied to determine relative levels ofresistance (105, 420, 1680, or 3360 g ae/ha). Table 15 shows thecomparisons drawn to dgt-1.

All glyphosate herbicide applications were made by track sprayer in a187 L/ha spray volume. Glyphosate used was of the commercial Durangodimethylamine salt formulation (480 g ae/L, Dow AgroSciences, LLC). Lowcopy T₁ plants that exhibited tolerance to either glufosinate orglyphosate were further accessed in the T₂ generation.

The first Arabidopsis transformations were conducted using dgt-31,dgt-32, and dgt-33 v1. T₁ transformants were first selected from thebackground of untransformed seed using a glufosinate selection scheme.Three flats or 30,000 seed were analyzed for each T₁ construct.Transformation frequency was calculated and results of T1 dgt-31,dgt-32, and dgt-33 constructs are listed in Table 15.

TABLE 15 Transformation frequency of T1 dgt-31, dgt-32, and dgt-33Arabidopsis constructs selected with glufosinate for selection of theselectable marker gene DSM-2. Transformation Construct CassetteFrequency (%) pDAB107532 AtUbi10/TraP14 dgt-32 v1 0.47 pDAB107533AtUbi10/TraP23 dgt-31 v1 0.36 pDAB107534 AtUbi10/TraP24 dgt-33 v1 0.68

T₁ plants selected above were subsequently transplanted to individualpots and sprayed with various rates of commercial glyphosate. Table 16compares the response of dgt-31, dgt-32, and dgt-33 v1 and control genesto impart glyphosate resistance to Arabidopsis T₁ transformants.Response is presented in terms of % visual injury 2 WAT. Data arepresented as a histogram of individuals exhibiting little or no injury(<20%), moderate injury (20-40%), or severe injury (>40%). An arithmeticmean and standard deviation is presented for each treatment. The rangein individual response is also indicated in the last column for eachrate and transformation. Wild-type non-transformed Arabidopsis (cv.Columbia) served as a glyphosate sensitive control. The DGT-31 (v1) genewith transit peptide TraP23 imparted slight herbicide tolerance toindividual T₁ Arabidopsis plants compared to the negative control, butthe gene exhibited improved tolerance with transit peptide TraP8. BothDGT-32 and DGT-33 demonstrated robust tolerance to glyphosate at therates tested with TraP8 and with their respective differing chloroplasttransit peptide (TraP14 and TraP24 respectively). Within a giventreatment, the level of plant response varied greatly, which can beattributed to the fact each plant represents an independenttransformation event and thus the copy number of the gene of interestvaries from plant to plant. Of important note, at each glyphosate ratetested, there were individuals that were more tolerant than others. Anoverall population injury average by rate is presented in Table 16 todemonstrate the significant difference between the plants transformedwith dgt-31, dgt-32, and dgt-33 v1 versus the dgt-1 v1 or Wild-typecontrols.

TABLE 16 dgt-31, dgt-32, and dgt-33 v1 transformed T₁ Arabidopsisresponse to a range of glyphosate rates applied postemergence, comparedto a dgt-1 (T4) homozygous resistant population, or a non-transformedcontrol. Visual % injury 2 weeks after treatment. % Injury % Injury 20-Std. Range Averages <20% 40% >40% Ave Dev. (%) TraP23 dgt-31 0 g ae/haglyphosate 4 0 0 0.0 0.0 0 105 g ae/ha 0 0 4 81.3 2.5 80-85  420 g ae/ha0 0 4 97.3 4.9 90-100 1680 g ae/ha 0 0 4 90.0 7.1 85-100 3360 g ae/ha 00 4 91.3 6.3 85-100 TraP14 dgt-32 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0105 g ae/ha 4 0 0 0.0 0.0 0 420 g ae/ha 2 0 2 30.0 29.4 0-60 1680 gae/ha 3 0 1 17.5 21.8 5-50 3360 g ae/ha 0 3 1 35.0 30.0 20-80  TraP24dgt-33 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha 2 2 0 21.3 14.95-40 420 g ae/ha 1 1 2 46.3 30.9 5-70 1680 g ae/ha 1 0 3 62.5 38.8 5-903360 g ae/ha 1 0 3 62.0 36.0 8-80 TraP8 dgt-31 0 g ae/ha glyphosate 4 00 0.0 0.0 0.0 105 g ae/ha glyphosate 0 1 3 0.0 43.8 17.0 420 g ae/haglyphosate 1 2 1 0.0 43.8 32.5 1680 g ae/ha glyphosate 0 1 3 0.0 71.327.8 3360 g ae/ha glyphosate 0 0 4 0.0 81.3 8.5 TraP8 dgt-32 0 g ae/haglyphosate 4 0 4 0.0 0.0 0.0 105 g ae/ha glyphosate 4 0 0 0.0 0.0 0.0420 g ae/ha glyphosate 4 0 0 0.0 7.5 5.0 1680 g ae/ha glyphosate 3 1 00.0 10.8 9.6 3360 g ae/ha glyphosate 4 0 0 0.0 12.8 3.2 TraP8 dgt-33 0 gae/ha glyphosate 4 0 0 0.0 0.0 0.0 105 g ae/ha glyphosate 4 0 0 0.0 0.00.0 420 g ae/ha glyphosate 4 0 0 0.0 2.5 3.8 1680 g ae/ha glyphosate 4 00 0.0 6.3 2.5 3360 g ae/ha glyphosate 3 1 0 0.0 20.0 13.5 dgt-1(transformed control 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha 01 3 42.5 15.0 20-50  420 g ae/ha 0 2 2 38.8 11.1 25-50  1680 g ae/ha 0 04 79.0 19.4 50-90  3360 g ae/ha 0 0 4 50.0 0.0 50 WT (non-transformedcontrol) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha 0 0 4 85.0 0.085 420 g ae/ha 0 0 4 100.0 0.0 100 1680 g ae/ha 0 0 4 100.0 0.0 100 3360g ae/ha 0 0 4 100.0 0.0 100

Maize Transformation.

Standard cloning methods, as described above, were used in theconstruction of binary vectors for use in Agrobacteriumtumefaciens-mediated transformation of maize. Table 17 lists the vectorswhich were constructed for maize transformation. The following geneelements were used in the vectors which contained dgt-28; the Zea maysUbiquitin 1 promoter (ZmUbi1; U.S. Pat. No. 5,510,474) was used to drivethe dgt-28 coding sequence which is flanked by a Zea mays Lipase 3′untranslated region (ZmLip 3′UTR; U.S. Pat. No. 7,179,902), theselectable marker cassette consists of the Zea mays Ubiquitin 1 promoterwhich was used to drive the aad-1 coding sequence (U.S. Pat. No.7,838,733) which is flanked by a Zea mays Lipase 3′ untranslated region.The aad-1 coding sequence confers tolerance to the phenoxy auxinherbicides, such as, 2,4-dichlorophenoxyacetic acid (2,4-D) and toaryloxyphenoxypropionate (AOPP) herbicides.

The dgt-28 constructs were built as standard binary vectors andAgrobacterium superbinary system vectors (Japan Tobacco, Tokyo, JP). Thestandard binary vectors include; pDAB107663, pDAB107664, pDAB107665, andpDAB107665. The Agrobacterium superbinary system vectors includepDAB108384, pDAB108385, pDAB108386, and pDAB108387.

Additional constructs were completed which contain a yellow fluorescentprotein (yfp); US Patent Application 2007/0298412) reporter gene.pDAB109812 contains a yfp reporter gene cassette which is driven by theZea mays Ubiquitin 1 promoter and flanked by the Zea mays per 5 3′untranslated region (Zm per5 3′UTR; U.S. Pat. No. 7,179,902), theselectable marker cassette consists of the sugar cane bacilliform viruspromoter (SCBV; U.S. Pat. No. 5,994,123) which is used to drive theexpression of aad-1 and is flanked by the Zea mays Lipase 3′untranslated region. pDAB101556 contains a yfp cassette which is drivenby the Zea mays Ubiquitin 1 promoter and flanked by the Zea mays per 53′ untranslated region, the selectable marker cassette consists of theZea mays Ubiquitin 1 promoter which is used to drive the expression ofaad-1 and is flanked by the Zea mays Lipase 3′ untranslated region.pDAB107698 contains a dgt-28 cassette which is driven by the Zea maysUbiquitin 1 promoter and is flanked by a Zea mays Lipase 3′ untranslatedregion, an yfp cassette which is driven by the Zea mays Ubiquitin 1promoter and flanked by the Zea mays per 5 3′ untranslated region, theselectable marker cassette consists of the sugar cane bacilliform viruspromoter which is used to drive the expression of aad-1 and is flankedby the Zea mays Lipase 3′ untranslated region. All three of theseconstructs are standard binary vectors.

TABLE 17 Maize Transformation Vectors Plasmid FIG. No. No: Descriptionof Gene Elements pDAB107663 35 ZmUbi1/TraP4 dgt-28/ZmLip 3′UTR ::ZmUbi1/aad-1/ZmLip 3′UTR binary vector pDAB107664 36 ZmUbi1/TraP8dgt-28/ZmLip 3′UTR :: ZmUbi1/aad-1/ZmLip 3′UTR binary vector pDAB10766537 ZmUbi1/TraP23 dgt-28/ZmLip 3′UTR :: ZmUbi1/aad-1/ZmLip 3′UTR binaryvector pDAB107666 38 ZmUbi1/TraP5 dgt-28/ZmLip 3′UTR ::ZmUbi1/aad-1/ZmLip 3′UTR binary vector pDAB109812 39 ZmUbi1/yfp/ZmPer53′UTR :: SCBV/aad-1/ZmLip 3′UTR binary vector pDAB101556 40ZmUbi1/yfp/ZmPer5 3′UTR :: ZmUbi1/aad-1/ZmLip 3′UTR binary vectorpDAB107698 41 ZmUbi1/TraP8 dgt-28/ZmLip 3′UTR :: ZmUbi1/yfp/ZmLip3′UTR::SCBV/aad-1/ZmLip 3′UTR pDAB108384 42 ZmUbi1/TraP4 dgt-28/ZmLip3′UTR:: ZmUbi1/aad-1/ZmLip 3′UTR superbinary vector pDAB108385 43ZmUbi1/TraP8 dgt-28/ZmLip 3′UTR :: ZmUbi1/aad-1/ZmLip 3′UTR superbinaryprecursor pDAB108386 44 ZmUbi1/TraP23 dgt-28/ZmLip 3′UTR ::ZmUbi1/aad-1/ZmLip 3′UTR superbinary precursor pDAB108387 45ZmUbi1/TraP5 dgt-28/ZmLip 3′UTR::ZmUbi1/aad-1/ZmLip 3′UTR superbinaryprecursor

Ear Sterilization and Embryo Isolation.

To obtain maize immature embryos, plants of the Zea mays inbred lineB104 were grown in the greenhouse and were self or sib-pollinated toproduce ears. The ears were harvested approximately 9-12 dayspost-pollination. On the experimental day, ears were surface-sterilizedby immersion in a 20% solution of sodium hypochlorite (5%) and shakenfor 20-30 minutes, followed by three rinses in sterile water. Aftersterilization, immature zygotic embryos (1.5-2.4 mm) were asepticallydissected from each ear and randomly distributed into micro-centrifugetubes containing liquid infection media (LS Basal Medium, 4.43 gm/L; N6Vitamin Solution [1000×], 1.00 mL/L; L-proline, 700.0 mg/L; Sucrose,68.5 gm/L; D(+) Glucose, 36.0 gm/L; 10 mg/ml of 2,4-D, 150 μL/L). For agiven set of experiments, pooled embryos from three ears were used foreach transformation.

Agrobacterium Culture Initiation:

Glycerol stocks of Agrobacterium containing the binary transformationvectors described above were streaked on AB minimal medium platescontaining appropriate antibiotics and were grown at 20° C. for 3-4days. A single colony was picked and streaked onto YEP plates containingthe same antibiotics and was incubated at 28° C. for 1-2 days.

Agrobacterium Culture and Co-Cultivation.

Agrobacterium colonies were taken from the YEP plate, suspended in 10 mLof infection medium in a 50 mL disposable tube, and the cell density wasadjusted to OD₆₀₀ nm of 0.2-0.4 using a spectrophotometer. TheAgrobacterium cultures were placed on a rotary shaker at 125 rpm, roomtemperature, while embryo dissection was performed. Immature zygoticembryos between 1.5-2.4 mm in size were isolated from the sterilizedmaize kernels and placed in 1 mL of the infection medium) and washedonce in the same medium. The Agrobacterium suspension (2 mL) was addedto each tube and the tubes were placed on a shaker platform for 10-15minutes. The embryos were transferred onto co-cultivation media (MSSalts, 4.33 gm/L; L-proline, 700.0 mg/L; Myo-inositol, 100.0 mg/L;Casein enzymatic hydrolysate 100.0 mg/L; 30 mM Dicamba-KOH, 3.3 mg/L;Sucrose, 30.0 gm/L; Gelzan™, 3.00 gm/L; Modified MS-Vitamin [1000×],1.00 ml/L; 8.5 mg/ml AgNo₃, 15.0 mg/L; DMSO, 100 μM), oriented with thescutellum facing up and incubated at 25° C., under 24-hour light at 50μmole m⁻² sec⁻¹ light intensity for 3 days.

Callus Selection and Regeneration of Putative Events.

Following the co-cultivation period, embryos were transferred to restingmedia (MS Salts, 4.33 gm/L; L-proline, 700.0 mg/L;1,2,3,5/4,6-Hexahydroxycyclohexane, 100 mg/L; MES[(2-(n-morpholino)-ethanesulfonic acid), free acid] 0.500 gm/L; Caseinenzymatic hydrolysate 100.0 mg/L; 30 mM Dicamba-KOH, 3.3 mg/L; Sucrose,30.0 gm/L; Gelzan 2.30 gm/L; Modified MS-Vitamin [1000×], 1.00 ml/L; 8.5mg/ml AgNo3, 15.0 mg/L; Carbenicillin, 250.0 mg/L) without selectiveagent and incubated under 24-hour light at 50 μmole m⁻² sec⁻¹ lightintensity and at 25° C. for 3 days.

Growth inhibition dosage response experiments suggested that glyphosateconcentrations of 0.25 mM and higher were sufficient to inhibit cellgrowth in the untransformed B104 maize line. Embryos were transferredonto Selection 1 media containing 0.5 mM glyphosate (MS Salts, 4.33gm/L; L-proline, 700.0 mg/L; Myo-inositol, 100.0 mg/L; MES[(2-(n-morpholino)-ethanesulfonic acid), free acid] 0.500 gm/L; Caseinenzymatic hydrolysate 100.0 mg/L; 30 mM Dicamba-KOH, 3.3 mg/L; Sucrose,30.0 gm/L; Gelzan™ 2.30 gm/L; Modified MS-Vitamin [1000×], 1.00 ml/L;8.5 mg/ml AgNo3, 15.0 mg/L; Carbenicillin, 250.0 mg/L) and incubated ineither dark and/or under 24-hour light at 50 μmole m⁻² sec⁻¹ lightintensity for 7-14 days at 28° C.

Proliferating embryogenic calli were transferred onto Selection 2 mediacontaining 1.0 mM glyphosate (MS Salts, 4.33 gm/L;1,2,3,5/4,6-Hexahydroxycyclohexane, 100 mg/L; L-proline, 700.0 mg/L; MES[(2-(n-morpholino)-ethanesulfonic acid), free acid] 0.500 gm/L; Caseinenzymatic hydrolysate 100.0 mg/L; 30 mM Dicamba-KOH, 3.3 mg/L; Sucrose,30.0 gm/L; Gelzan™ 2.30 gm/L; Modified MS-Vitamin [1000×], 1.00 ml/L;8.5 mg/mL AgNo3, 15.0 mg/L; Carbenicillin, 250.0 mg/L; R-Haloxyfop acid0.1810 mg/L), and were incubated in either dark and/or under 24-hourlight at 50 μmole m⁻² sec⁻¹ light intensity for 14 days at 28° C. Thisselection step allowed transgenic callus to further proliferate anddifferentiate. The callus selection period lasted for three to fourweeks.

Proliferating, embryogenic calli were transferred onto PreReg mediacontaining 0.5 mM glyphosate (MS Salts, 4.33 gm/L;1,2,3,5/4,6-Hexahydroxycyclohexane, 100 mg/L; L-proline, 350.0 mg/L; MES[(2-(n-morpholino)-ethanesulfonic acid), free acid] 0.250 gm/L; Caseinenzymatic hydrolysate 50.0 mg/L; NAA-NaOH 0.500 mg/L; ABA-EtOH 2.50mg/L; BA 1.00 mg/L; Sucrose, 45.0 gm/L; Gelzan™ 2.50 gm/L; ModifiedMS-Vitamin [1000×], 1.00 ml/L; 8.5 mg/ml AgNo3, 1.00 mg/L;Carbenicillin, 250.0 mg/L) and cultured under 24-hour light at 50 μmolem⁻² sec⁻¹ light intensity for 7 days at 28° C.

Embryogenic calli with shoot-like buds were transferred ontoRegeneration media containing 0.5 mM glyphosate (MS Salts, 4.33 gm/L;1,2,3,5/4,6-Hexahydroxycyclohexane, 100.0 mg/L; Sucrose, 60.0 gm/L;Gellan Gum G434™ 3.00 gm/L; Modified MS-Vitamin [1000×], 1.00 ml/L;Carbenicillin, 125.0 mg/L) and cultured under 24-hour light at 50 μmolem⁻² sec⁻¹ light intensity for 7 days.

Small shoots with primary roots were transferred to rooting media (MSSalts, 4.33 gm/L; Modified MS-Vitamin [1000×], 1.00 ml/L;1,2,3,5/4,6-Hexahydroxycyclohexane, 100 mg/L; Sucrose, 60.0 gm/L; GellanGum G434™ 3.00 gm/L; Carbenicillin, 250.0 mg/L) in phytotrays and wereincubated under 16/8 hr. light/dark at 140-190 μmole m⁻² sec⁻¹ lightintensity for 7 days at 27° C. Putative transgenic plantlets wereanalyzed for transgene copy number using the protocols described aboveand transferred to soil.

Molecular Confirmation of the Presence of the Dgt-28 and Aad-1Transgenes within Maize Plants.

The presence of the dgt-28 and aad-1 polynucleotide sequences wereconfirmed via hydrolysis probe assays. Isolated T₀ Maize plants wereinitially screened via a hydrolysis probe assay, analogous to TAQMAN™,to confirm the presence of a aad-1 and dgt-28 transgenes. The datagenerated from these studies were used to determine the transgene copynumber and used to select transgenic maize events for back crossing andadvancement to the T₁ generation.

Tissue samples were collected in 96-well plates, tissue maceration wasperformed with a KLECO™ tissue pulverizer and stainless steel beads(Hoover Precision Products, Cumming, Ga.), in Qiagen™ RLT buffer.Following tissue maceration, the genomic DNA was isolated inhigh-throughput format using the Biosprint 96™ Plant kit (Qiagen,Germantown, Md.) according to the manufacturer's suggested protocol.Genomic DNA was quantified by Quant-IT™ Pico Green DNA assay kit(Molecular Probes, Invitrogen, Carlsbad, Calif.). Quantified genomic DNAwas adjusted to around 2 ng/μL for the hydrolysis probe assay using aBIOROBOT3000™ automated liquid handler (Qiagen, Germantown, Md.).Transgene copy number determination by hydrolysis probe assay, analogousto TAQMAN® assay, was performed by real-time PCR using the LIGHTCYCLER®480 system (Roche Applied Science, Indianapolis, Ind.). Assays weredesigned for aad-1, dgt-28 and an internal reference gene Invertase(Genbank Accession No: U16123.1) using the LIGHTCYCLER® Probe DesignSoftware 2.0. For amplification, LIGHTCYCLER®480 Probes Master mix(Roche Applied Science, Indianapolis, Ind.) was prepared at 1× finalconcentration in a 10 μL volume multiplex reaction containing 0.4 μM ofeach primer for aad-1 and dgt-28 and 0.2 μM of each probe (Table 18).

A two-step amplification reaction was performed with an extension at 60°C. for 40 seconds with fluorescence acquisition. All samples were runand the averaged Cycle threshold (Ct) values were used for analysis ofeach sample. Analysis of real time PCR data was performed usingLightCycler® software release 1.5 using the relative quant module and isbased on the ΔΔCt method. Controls included a sample of genomic DNA froma single copy calibrator and known two copy check that were included ineach run. Table 19 lists the results of the hydrolysis probe assays.

TABLE 18 Primer and probe sequences used for hydrolysisprobe assay of aad-1, dgt-28 and internal reference (Invertase).Oligonu- SEQ cleotide Gene ID Name Detected NO: Oligo Sequence GAAD1Faad-1 58 TGTTCGGTTCCCTCTACCAA forward primer GAAD1P aad-1 59CACAGAACCGTCGCTTCAGCAACA probe GAAD1R aad-1 60 CAACATCCATCACCTTGACTGAreverse primer IV-Probe Invertase 61 CGAGCAGACCGCCGTGTACTTCTACC probeIVF-Taq Invertase 62 TGGCGGACGACGACTTGT forward primer IVR-Taq Invertase63 AAAGTTTGGAGGCTGCCGT reverse primer zmDGT28  dgt-28 64TTCAGCACCCGTCAGAAT F forward primer zmDGT28  dgt-28 65TGCCGAGAACTTGAGGAGGT FAM probe zmDGT28  dgt-28 66 TGGTCGCCATAGCTTGT Rreverse primer

TABLE 19 T₀ copy amount results for dgt-28 events. Low copy eventsconsisted of 1-2 transgene copies, single copy numbers are listed inparenthesis. High copy events contained 3 or more transgene copies.Plasmid used for # of Low Copy # of High Copy Transformation Events(single copy) Events pDAB107663 43 (31) 10 pDAB107664 30 (24) 5pDAB107665 40 (27) 10 pDAB107666 24 (12) 12 pDAB109812 2 (1) 0pDAB101556 25 (15) 10 pDAB107698 3 (1) 2

Herbicide Tolerance in Dgt-28 Transformed Corn.

Zea mays dgt-28 transformation events (T₀) were allowed to acclimate inthe greenhouse and were grown until plants had transitioned from tissueculture to greenhouse growing conditions (i.e., 2-4 new, normal lookingleaves had emerged from the whorl). Plants were grown at 27° C. under 16hour light:8 hour dark conditions in the greenhouse. The plants werethen treated with commercial formulations of DURANGO DMA™ (containingthe herbicide glyphosate) with the addition of 2% w/v ammonium-sulfate.Herbicide applications were made with a track sprayer at a spray volumeof 187 L/ha, 50-cm spray height. T₀ plants were sprayed with a range ofglyphosate from 280-4480 g ae/ha glyphosate, which is capable ofsignificant injury to untransformed corn lines. A lethal dose is definedas the rate that causes >95% injury to the B104 inbred.

The results of the T₀ dgt-28 corn plants demonstrated that tolerance toglyphosate was achieved at rates up to 4480 g ae/ha. A specific mediatype was used in the T₀ generation. Minimal stunting and overall plantgrowth of transformed plants compared to the non-transformed controlsdemonstrated that dgt-28 provides robust tolerance to glyphosate whenlinked to the TraP5, TraP8, and TraP23 chloroplast transit peptides.

Selected T₀ plants are selfed or backcrossed for furthercharacterization in the next generation. 100 chosen dgt-28 linescontaining the T₁ plants are sprayed with 140-1120 g ae/ha glufosinateor 105-1680 g ae/ha glyphosate. Both the selectable marker andglyphosate resistant gene are constructed on the same plasmid.Therefore, if one herbicide tolerant gene is selected for by sprayingwith an herbicide, both genes are believed to be present. At 14 DAT,resistant and sensitive plants are counted to determine the percentageof lines that segregated as a single locus, dominant Mendelian trait(3R:1S) as determined by Chi square analysis. These data demonstratethat dgt-28 is inheritable as a robust glyphosate resistance gene in amonocot species. Increased rates of glyphosate are applied to the T₁ orF₁ survivors to further characterize the tolerance and protection thatis provided by the dgt-28 gene.

Post-Emergence Herbicide Tolerance in Dgt-28 Transformed T₀ Corn.

To events of dgt-28 linked with TraP4, TraP5, TraP8 and TraP23 weregenerated by Agrobacterium transformation and were allowed to acclimateunder controlled growth chamber conditions until 2-4 new, normal lookingleaves had emerged from the whorl. Plants were assigned individualidentification numbers and sampled for copy number analyses of bothdgt-28 and aad-1. Based on copy number analyses, plants were selectedfor protein expression analyses. Plants were transplanted into largerpots with new growing media and grown at 27° C. under 16 hour light:8hour dark conditions in the greenhouse. Remaining plants that were notsampled for protein expression were then treated with commercialformulations of DURANGO DMA™ (glyphosate) with the addition of 2% w/vammonium-sulfate. Treatments were distributed so that each grouping ofplants contained T₀ events of varying copy number. Herbicideapplications were made with a track sprayer at a spray volume of 187L/ha, 50-cm spray height. T₀ plants were sprayed with a range ofglyphosate from 280-4480 g ae/ha glyphosate capable of significantinjury to untransformed corn lines. A lethal dose is defined as the ratethat causes >95% injury to the B104 inbred. B104 was the geneticbackground of the transformants.

Results of T₀ dgt-28 corn plants demonstrate that tolerance toglyphosate was achieved up to 4480 g ae/ha. Table 20. Minimal stuntingand overall plant growth of transformed plants compared to thenon-transformed controls demonstrated that dgt-28 provides robustprotection to glyphosate when linked to TraP5, TraP8, and TraP23.

TABLE 20 Response of T₀ dgt-28 events of varying copy numbers to ratesof glyphosate ranging from 280-4480 g ae/ha + 2.0% w/v ammonium sulfate14 days after treatment. % Injury % Injury 20- Std. Range ApplicationRate <20% 40% >40% Ave Dev. (%) TraP4 dgt-28 0 g ae/ha glyphosate 4 0 00.0 0.0 0 280 g ae/ha 5 0 0 1.0 2.2 0-5  560 g ae/ha 6 0 0 2.0 4.0 0-101120 g ae/ha 12 0 0 1.3 3.1 0-10 2240 g ae/ha 7 0 0 1.7 4.5 0-12 4480 gae/ha 7 0 0 1.1 3.0 0-8  TraP8 dgt-28 0 g ae/ha glyphosate 6 0 0 0.0 0.00 280 g ae/ha 5 1 0 6.7 8.8 0-20 560 g ae/ha 0 2 0 20.0 0.0 20  1120 gae/ha 7 0 0 1.4 2.4 0-5  2240 g ae/ha 3 1 0 7.5 15.0 0-30 4480 g ae/ha 60 0 1.7 4.1 0-10 TraP23 dgt-28 0 g ae/ha glyphosate 6 0 0 0.8 2.0 0-5 280 g ae/ha 7 0 0 0.0 0.0 0 560 g ae/ha 4 0 0 1.3 2.5 0-5  1120 g ae/ha10 2 0 3.3 7.8 0-20 2240 g ae/ha 6 0 0 1.3 3.3 0-8  4480 g ae/ha 6 1 04.3 7.9 0-20 TraP5 dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 280 gae/ha 7 1 0 5.0 14.1 0-40 560 g ae/ha 8 0 0 0.6 1.8 0-5  1120 g ae/ha 71 0 5.0 14.1 0-40 2240 g ae/ha 8 0 0 0.0 0.0 0 4480 g ae/ha 8 0 0 0.00.0 0

Protein expression analyses by standard ELISA demonstrated a mean rangeof DGT-28 protein from 12.6-22.5 ng/cm² across the constructs tested.

Confirmation of Glyphosate Tolerance in the F₁ Generation UnderGreenhouse Conditions.

Single copy T₀ plants that were not sprayed were backcrossed to thenon-transformed background B104 for further characterization in the nextgeneration. In the T₁ generation, glyphosate tolerance was assessed toconfirm the inheritance of the dgt-28 gene. For T₁ plants, the herbicideASSURE II™ (35 g ae/ha quizalofop-methyl) was applied at the V1 growthstage to select for the AAD-1 protein. Both the selectable marker andglyphosate resistant gene are constructed on the same plasmid. Thereforeif one gene is selected, both genes are believed to be present. After 7DAT, resistant and sensitive plants were counted and null plants wereremoved from the population. These data demonstrate that dgt-28 (v1) isheritable as a robust glyphosate resistance gene in a monocot species.Plants were sampled for characterization of DGT-28 protein by standardELISA and RNA transcript level. Resistant plants were sprayed with560-4480 g ae/ha glyphosate as previously described. The datademonstrate robust tolerance of dgt-28 linked with the chloroplasttransit peptides TraP4, TraP5, TraP8 and TraP23 up to 4480 g ae/haglyphosate. Table 21.

TABLE 21 Response of F₁ single copy dgt-28 events to rates of glyphosateranging from 560-4480 g ae/ha + 2.0% w/v ammonium sulfate 14 days aftertreatment. % Injury % Injury 20- Std. Range Application Rate <20%40% >40% Ave Dev. (%) B104/TraP4::dgt-28 0 g ae/ha glyphosate 4 0 0 0.00.0 0 560 g ae/ha 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0 0 9.0 1.2  8-10 2240g ae/ha 4 0 0 2.5 2.9 0-5 4480 g ae/ha 4 0 0 0.0 0.0 0B104/TraP8::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 4 00 1.3 2.5 0-5 1120 g ae/ha 4 0 0 0.0 0.0 0 2240 g ae/ha 4 0 0 5.0 4.1 0-10 4480 g ae/ha 4 0 0 6.3 2.5  5-10 B104/TraP23::dgt-28 0 g ae/haglyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 3 1 0 10.0 10.0  5-25 1120 gae/ha 2 2 0 18.8 11.8 10-35 2240 g ae/ha 4 0 0 12.5 2.9 10-15 4480 gae/ha 3 1 0 10.0 7.1  5-20 B104/TraP5::dgt-28 0 g ae/ha glyphosate 4 0 00.0 0.0 0 560 g ae/ha 4 0 0 8.0 0.0 8 1120 g ae/ha 4 0 0 11.3 3.0  8-152240 g ae/ha 4 0 0 12.5 2.9 10-15 4480 g ae/ha 4 0 0 10.0 2.5 10-15Non-transformed B104 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 00 4 100.0 0.0 100  1120 g ae/ha 0 0 4 100.0 0.0 100  2240 g ae/ha 0 0 4100.0 0.0 100  4480 g ae/ha 0 0 4 100.0 0.0 100 

Protein expression data demonstrate a range of mean DGT-28 protein from42.2-88.2 ng/cm² across T₁ events and constructs tested, establishingprotein expression in the T₁ generation.

Characterization of dgt-28 corn under field conditions. Single copy T₁events were sent to a field location to create both hybrid hemizygousand inbred homozygous seed for additional characterization. Hybrid seedswere created by crossing T₁ events in the maize transformation line B104to the inbred line 4XP811 generating hybrid populations segregating 1:1(hemizygous:null) for the event. The resulting seeds were shipped to 2separate locations. A total of five single copy events per constructwere planted at each location in a randomized complete block design intriplicate. The fields were designed for glyphosate applications tooccur at the V4 growth stage and a separate grouping of plants to beapplied at the V8 growth stage. The 4XP811/B104 conventional hybrid wasused as a negative control.

Experimental rows were treated with 184 g ae/ha ASSURE II™ (106 g ai/Lquizalofop-methyl) to eliminate null segregants. All experimentalentries segregated 1:1 (sensitive:resistant) (p=0.05) with respect tothe ASSURE II™ application. Selected resistant plants were sampled fromeach event for quantification of the DGT-28 protein by standard ELISA.

Quizalofop-methyl resistant plants were treated with the commercialherbicide DURANGO DMA™ (480 g ae/L glyphosate) with the addition of 2.5%w/v ammonium-sulfate at either the V4 or V8 growth stages. Herbicideapplications were made with a boom sprayer calibrated to deliver avolume of 187 L/ha, 50-cm spray height. Plants were sprayed with a rangeof glyphosate from 1120-4480 g ae/ha glyphosate, capable of significantinjury to untransformed corn lines. A lethal dose is defined as the ratethat causes >95% injury to the 4XP811 inbred. Visual injury assessmentswere taken for the percentage of visual chlorosis, percentage ofnecrosis, percentage of growth inhibition and total visual injury at 7,14 and 21 DAT (days after treatment). Assessments were compared to theuntreated checks for each line and the negative controls.

Visual injury data for all assessment timings demonstrated robusttolerance up to 4480 g ae/ha DURANGO DMA™ at both locations andapplication timings. Representative events for the V4 application arepresented from one location and are consistent with other events,application timings and locations. Table 22. One event from theconstruct containing dgt-28 linked with TraP23 (pDAB107665) was tolerantto the ASSURE II™ selection for the AAD-1 protein, but was sensitive toall rates of glyphosate applied.

TABLE 22 Response of dgt-28 events applied with a range of glyphosatefrom 1120-4480 g ae/ha + 2.5% w/v ammonium sulfate at the V4 growthstage. % Injury % Injury 20- Std. Range Application Rate <20% 40% >40%Ave Dev. (%) 4XPB11//B104/ TraP4::dgt-28 0 g ae/ha glyphosate 4 0 0 0.00.0 0 1120 g ae/ha 4 0 0 0.0 0.0 0 2240 g ae/ha 4 0 0 0.0 0.0 0 4480 gae/ha 4 0 0 0.0 0.0 0 4XPB11//B104/ TraP8::dgt-28 0 g ae/ha glyphosate 40 0 0.0 0.0 0 1120 g ae/ha 4 0 0 0.0 0.0 0 2240 g ae/ha 4 0 0 0.0 0.0 04480 g ae/ha 4 0 0 0.0 0.0 0 4XPB11//B104/ TraP23::dgt-28 0 g ae/haglyphosate 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0 0 0.0 0.0 0 2240 g ae/ha 4 00 0.0 0.0 0 4480 g ae/ha 4 0 0 0.0 0.0 0 4XPB11//B104/ TraP5::dgt-28 0 gae/ha glyphosate 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0 0 0.0 0.0 0 2240 gae/ha 4 0 0 0.0 0.0 0 4480 g ae/ha 4 0 0 0.0 0.0 0 Non-transformed4XPB11//B104 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 1120 g ae/ha 0 0 4100.0 0.0 100 2240 g ae/ha 0 0 4 100.0 0.0 100 4480 g ae/ha 0 0 4 100.00.0 100

Additional assessments were made during the reproductive growth stagefor the 4480 g ae/ha glyphosate rate. Visual assessments of tassels,pollination timing and ear fill were similar to the untreated checks ofeach line for all constructs, application timings and locations.Quantification results for the DGT-28 protein demonstrated a range ofmean protein expression from 186.4-303.0 ng/cm². Data demonstratesrobust tolerance of dgt-28 transformed corn under field conditionsthrough the reproductive growth stages up to 4480 g ae/ha glyphosate.Data also demonstrated DGT-28 protein detection and function based onspray tolerance results.

Confirmation of Heritability and Tolerance of Dgt-28 Corn in theHomozygous State.

Seed from the T₁S2 were planted under greenhouse conditions aspreviously described. The same five single copy lines that werecharacterized under field conditions were characterized in thehomogeneous state. Plants were grown until the V3 growth stage andseparated into three rates of glyphosate ranging from 1120-4480 g ae/haglyphosate (DURANGO DMA™) and four replicates per treatment.Applications were made in a track sprayer as previously described andwere formulated in 2.0% w/v ammonium sulfate. An application of ammoniumsulfate served as an untreated check for each line. Visual assessmentswere taken 7 and 14 days after treatment as previously described. Datademonstrated robust tolerance up to 4480 g ae/ha glyphosate for allevents tested. Table 23.

TABLE 23 Response of homozygous dgt-28 events applied with a range ofglyphosate from 1120-4480 g ae/ha + 2.0% w/v ammonium sulfate. % Injury% Injury 20- Std. Range Application Rate <20% 40% >40% Ave Dev. (%)TraP4::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0 00.0 0.0 0 2240 g ae/ha 4 0 0 3.8 2.5 0-5 4480 g ae/ha 4 0 0 14.3 1.512-15 TraP8::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 1120 g ae/ha 40 0 0.0 0.0 0 2240 g ae/ha 4 0 0 9.0 1.2  8-10 4480 g ae/ha 4 0 0 11.32.5 10-15 TraP23::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 1120 gae/ha 4 0 0 4.5 3.3 0-8 2240 g ae/ha 4 0 0 7.5 2.9  5-10 4480 g ae/ha 40 0 15.0 0.0 15  TraP5::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 1120g ae/ha 4 0 0 1.3 2.5 0-5 2240 g ae/ha 4 0 0 9.0 2.0  8-12 4480 g ae/ha4 0 0 15.0 2.4 12-18 Non-transformed B104 0 g ae/ha glyphosate 4 0 0 0.00.0 0 1120 g ae/ha 0 0 4 100.0 0.0 100  2240 g ae/ha 0 0 4 100.0 0.0100  4480 g ae/ha 0 0 4 100.0 0.0 100 

The line from pDAB107665 that was not tolerant under field conditionsdemonstrated no tolerance to glyphosate and therefore consistent withfield observations (data not shown). With the exception of the one linepreviously mentioned, all replicates that were treated with glyphosatefrom the lines were not sensitive to glyphosate. Therefore datademonstrates heritability to a homogeneous population of dgt-28 corn ina Mendelian fashion. Expression of the DGT-28 protein by standard ELISAdemonstrated a range of mean protein expression from 27.5-65.8 ng/cm²across single copy events that were tolerant to glyphosate. Datademonstrates functional protein and stability of the DGT-28 proteinacross generations.

Postemergence Herbicide Tolerance Use of Glyphosate as a SelectableMarker.

As previously described, To transformed plants were moved from tissueculture and acclimated in the greenhouse. The events tested containeddgt-28 linked to TraP5, TraP8, and TraP23 chloroplast transit peptides.It was demonstrated that these T₀ plants provided robust tolerance up to4480 g ae/ha glyphosate, and non-transformed plants were controlled withglyphosate at concentrations as low as 280 g ae/ha. These datademonstrate that dgt-28 can be utilized as a selectable marker using aconcentration of glyphosate ranging from 280-4480 g ae/ha.

A number of seed from fixed lines of corn which contain the dgt-28transgene are spiked into a number of non-transformed corn seed. Theseed are planted and allowed to grow to the V1-V3 developmental stage,at which time the plantlets are sprayed with a selecting dose ofglyphosate in the range of 280-4480 g ae/ha. Following 7-10 days,sensitive and resistant plants are counted, and the amount of glyphosatetolerant plants correlates with the original number of transgenic seedcontaining the dgt-28 transgene which are planted.

Stacking of Dgt-28 Corn.

The AAD-1 protein is used as the selectable marker in dgt-28 transformedcorn for research purposes. The aad-1 gene can also be utilized as aherbicide tolerant trait in corn to provide robust 2,4-D tolerance up toa V8 application in a crop. Four events from the constructs pDAB107663(TraP4::dgt-28), pDAB107664 (TraP8::dgt-28) and pDAB107666(TraP5::dgt-28) were characterized for the tolerance of a tank mixapplication of glyphosate and 2,4-D. The characterization study wascompleted with F₁ seed under greenhouse conditions. Applications weremade in a track sprayer as previously described at the following rates:1120-2240 g ae/ha glyphosate (selective for the dgt-28 gene), 1120-2240g ae/ha 2,4-D (selective for the aad-1 gene), or a tank mixture of thetwo herbicides at the rates described. Plants were graded at 7 and 14DAT. Spray results for applications of the herbicides at 2240 g ae/haare shown in Table 24.

TABLE 24 Response of F₁ aad-1 and dgt-28 corn sprayed with 2240 g ae/haof 2,4-D, glyphosate and a tank mix combination of the two herbicides 14days after treatment. 2240 g ae/ha 2240 g ae/ha 2240 g ae/ha 2,4-D +2,4-D glyphosate 2240 g ae/ha glyphosate Mean Mean Mean % Std. % Std. %Std. F₁ Event injury Dev. injury Dev. injury Dev. 107663[3]- 5.0 4.1 3.84.8 8.8 3.0 012.AJ001 107663[3]- 2.5 5.0 1.3 2.5 5.0 5.8 029.AJ001107663[3]- 2.5 2.9 11.8 2.9 13.8 2.5 027.AJ001 107663[3]- 3.8 2.5 11.51.0 12.8 1.5 011.AJ001 B104 27.5 17.7 100.0 0.0 100.0 0.0

The results confirm that dgt-28 can be successfully stacked with aad-1,thus increasing the spectrum herbicides that may be applied to the cropof interest (glyphosate+phenoxyacetic acids for dgt-28 and aad-1,respectively). In crop production where hard to control broadleaf weedsor resistant weed biotypes exist the stack can be used as a means ofweed control and protection of the crop of interest. Additional input oroutput traits can also be stacked with the dgt-28 gene in corn and otherplants.

Soybean Transformation.

Transgenic soybean (Glycine max) containing a stably integrated dgt-28transgene was generated through Agrobacterium-mediated transformation ofsoybean cotyledonary node explants. A disarmed Agrobacterium straincarrying a binary vector containing a functional dgt-28 was used toinitiate transformation.

Agrobacterium-mediated transformation was carried out using a modifiedhalf-cotyledonary node procedure of Zeng et al. (Zeng P., Vadnais D. A.,Zhang Z., Polacco J. C., (2004), Plant Cell Rep., 22(7): 478-482).Briefly, soybean seeds (cv. Maverick) were germinated on basal media andcotyledonary nodes are isolated and infected with Agrobacterium. Shootinitiation, shoot elongation, and rooting media are supplemented withcefotaxime, timentin and vancomycin for removal of Agrobacterium.Selection via a herbicide was employed to inhibit the growth ofnon-transformed shoots. Selected shoots are transferred to rootingmedium for root development and then transferred to soil mix foracclimatization of plantlets.

Terminal leaflets of selected plantlets were treated topically (leafpaint technique) with a herbicide to screen for putative transformants.The screened plantlets were transferred to the greenhouse, allowed toacclimate and then leaf-painted with a herbicide to reconfirm tolerance.These putative transformed T₀ plants were sampled and molecular analyseswas used to confirm the presence of the herbicidal selectable marker,and the dgt-28 transgene. T₀ plants were allowed to self fertilize inthe greenhouse to produce T₁ seed.

A second soybean transformation method can be used to produce additionaltransgenic soybean plants. A disarmed Agrobacterium strain carrying abinary vector containing a functional dgt-28 is used to initiatetransformation.

Agrobacterium-mediated transformation was carried out using a modifiedhalf-seed procedure of Paz et al., (Paz M., Martinez J., Kalvig A.,Fonger T., and Wang K., (2005) Plant Cell Rep., 25: 206-213). Briefly,mature soybean seeds were sterilized overnight with chlorine gas andimbibed with sterile H₂O twenty hours before Agrobacterium-mediatedplant transformation. Seeds were cut in half by a longitudinal cut alongthe hilum to separate the seed and remove the seed coat. The embryonicaxis was excised and any axial shoots/buds were removed from thecotyledonary node. The resulting half seed explants were infected withAgrobacterium. Shoot initiation, shoot elongation, and rooting mediawere supplemented with cefotaxime, timentin and vancomycin for removalof Agrobacterium. Herbicidal selection was employed to inhibit thegrowth of non-transformed shoots. Selected shoots were transferred torooting medium for root development and then transferred to soil mix foracclimatization of plantlets.

Putative transformed T₀ plants were sampled and molecular analyses wasused to confirm the presence of the selectable marker and the dgt-28transgene. Several events were identified as containing the transgenes.These T₀ plants were advanced for further analysis and allowed to selffertilize in the greenhouse to give rise to T₁ seed.

Confirmation of Heritability of Dgt-28 to the T₁ Generation.

Heritability of the DGT-28 protein into T₁ generation was assessed inone of two ways. The first method included planting T₁ seed intoMetro-mix media and applying 411 g ae/ha IGNITE™ 280 SL on germinatedplants at the 1^(st) trifoliate growth stage. The second methodconsisted of homogenizing seed for a total of 8 replicates using a ballbearing and a genogrinder. ELISA strip tests to detect for the PATprotein were then used to detect heritable events as the selectablemarker was on the same plasmid as dgt-28. For either method if a singleplant was tolerant to glufosinate or was detected with the PAT ELISAstrip test, the event demonstrated heritability to the T₁ generation.

A total of five constructs were screened for heritability as previouslydescribed. The plasmids contained dgt-28 linked with TraP4, TraP8 andTraP23 The events across constructs demonstrated 68% heritability of thePAT::DGT-28 protein to the T₁ generation.

Postemergence Herbicide Tolerance in Dgt-28 Transformed T₁ Soybean.

Seeds from T₁ events that were determined to be heritable by thepreviously described screening methods were planted in Metro-mix mediaunder greenhouse conditions. Plants were grown until the 1^(st)trifoliate was fully expanded and treated with 411 g ae/ha IGNITE™ 280SL for selection of the pat gene as previously described. Resistantplants from each event were given unique identifiers and sampled forzygosity analyses of the dgt-28 gene. Zygosity data were used to assign2 hemizygous and 2 homozygous replicates to each rate of glyphosateapplied allowing for a total of 4 replicates per treatment when enoughplants existed. These plants were compared against wildtype Petitehavana tobacco. All plants were sprayed with a track sprayer set at 187L/ha. The plants were sprayed from a range of 560-4480 g ae/ha DURANGO™dimethylamine salt (DMA). All applications were formulated in water withthe addition of 2% w/v ammonium sulfate (AMS). Plants were evaluated at7 and 14 days after treatment. Plants were assigned an injury ratingwith respect to overall visual stunting, chlorosis, and necrosis. The T₁generation is segregating, so some variable response is expected due todifference in zygosity.

TABLE 25 Spray results demonstrate at 14 DAT (days after treatment)robust tolerance up to 4480 g ae/ha glyphosate of at least one dgt-28event per construct characterized. Representative single copy events ofthe constructs all provided tolerance up to 4480 g ae/ha compared to theMaverick negative control. % Injury % Injury 20- Std. Range ApplicationRate <20% 40% >40% Ave Dev. (%) pDAB107543 (TraP4::dgt-28) 0 g ae/haglyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 0 4 0 33.8 7.5 25-40 1120 g ae/ha2 2 0 25.0 11.5 15-35 2240 g ae/ha 2 2 0 17.5 2.9 15-20 4480 g ae/ha 0 22 33.8 13.1 20-45 pDAB107545 (TraP8::dgt-28) 0 g ae/ha glyphosate 4 0 00.0 0.0 0 560 g ae/ha 4 0 0 1.5 1.0 0-2 1120 g ae/ha 4 0 0 2.8 1.5 2-52240 g ae/ha 4 0 0 5.0 2.4 2-8 4480 g ae/ha 4 0 0 9.5 1.9  8-12pDAB107548 (TraP4::dgt-28) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 gae/ha 4 0 0 1.8 2.4 0-5 1120 g ae/ha 4 0 0 2.8 1.5 2-5 2240 g ae/ha 4 00 3.5 1.7 2-5 4480 g ae/ha 4 0 0 8.8 3.0  5-12 pDAB107553(TraP23::dgt-28) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 4 0 05.0 0.0 5 1120 g ae/ha 4 0 0 9.0 1.2  8-10 2240 g ae/ha 4 0 0 10.5 1.010-12 4480 g ae/ha 4 0 0 16.5 1.7 15-18 Maverick (neg. control) 0 gae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 0 0 4 82.5 12.6  70-1001120 g ae/ha 0 0 4 100.0 0.0 100  2240 g ae/ha 0 0 4 100.0 0.0 100  4480g ae/ha 0 0 4 100.0 0.0 100 

Dgt-28 Protection Against Elevated Glyphosate Rates in the T₂Generation.

A 45 plant progeny test was conducted on two to five T₂ lines of dgt-28per construct. Homozygous lines were chosen based on zygosity analysescompleted in the previous generation. The seeds were planted aspreviously described. Plants were then sprayed with 411 g ae/ha IGNITE280 SL for the selection of the pat selectable marker as previouslydescribed. After 3 DAT, resistant and sensitive plants were counted.

For constructs containing TraP4 linked with dgt-28 (pDAB107543 andpDAB107548), nine out of twelve lines tested did not segregate, therebyconfirming homogeneous lines in the T₂ generation. Lines containingTraP8 linked with dgt-28 (pDAB107545) demonstrated two out of the fourlines with no segregants and demonstrating Mendelian inheritance throughat least two generation of dgt-28 in soybean. Tissue samples were takenfrom resistant plants and the DGT-28 protein was quantified by standardELISA methods. Data demonstrated a range of mean DGT-28 protein from32.8-107.5 ng/cm² for non-segregating T₂ lines tested. Lines from theconstruct pDAB107553 (TraP23::dgt-28) were not previously selected withglufosinate, and the dose response of glyphosate was utilized as both totest homogenosity and tolerance to elevated rates of glyphosate.Replicates from the lines from construct pDAB107553 were tolerant torates ranging from 560-4480 g ae/ha glyphosate, and were thereforeconfirmed to be a homogeneous population and heritable to at least twogenerations.

Rates of DURANGO DMA ranging from 560-4480 g ae/ha glyphosate wereapplied to 2-3 trifoliate soybean as previously described. Visual injurydata 14 DAT confirmed the tolerance results that were demonstrated inthe T₁ generation.

TABLE 26 The data demonstrate robust tolerance of the dgt-28 tobacco upto 3360 g ae/ha glyphosate through two generations, compared to thenon-transformed control. % Injury % Injury 20- Std. Range ApplicationRate <20% 40% >40% Ave Dev. (%) pDAB107543 (TraP4::dgt-28) 0 g ae/haglyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 4 0 0 8.0 0.0 8 1120 g ae/ha 4 00 14.3 1.5 12-15 2240 g ae/ha 4 0 0 18.0 0.0 18 4480 g ae/ha 0 4 0 24.53.3 20-28 pDAB107545 (TraP8::dgt-28) 0 g ae/ha glyphosate 4 0 0 0.0 0.00 560 g ae/ha 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0 0 2.8 1.5 2-5 2240 gae/ha 4 0 0 5.0 0.0 5 4480 g ae/ha 4 0 0 10.0 0.0 10 pDAB107548(TraP4::dgt-28) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 4 0 00.0 0.0 0 1120 g ae/ha 4 0 0 0.0 0.0 0 2240 g ae/ha 4 0 0 0.0 0.0 0 4480g ae/ha 4 0 0 10.0 0.0 10 pDAB107553 (TraP23::dgt-28) 0 g ae/haglyphosate 4 0 0 — 0.0 0.0 560 g ae/ha 4 0 0 — 10.0 0.0 1120 g ae/ha 4 00 — 10.0 −4.4 2240 g ae/ha 4 0 0 — 13.0 −2.4 4480 g ae/ha 3 1 0 — 15.54.1 Maverick (neg. control) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 gae/ha 0 0 4 77.5 15.0  70-100 1120 g ae/ha 0 0 4 97.5 2.9  95-100 2240 gae/ha 0 0 4 100.0 0.0 100 4480 g ae/ha 0 0 4 100.0 0.0 100

Transformation of Rice with Dgt-28.

Transgenic rice (Oryza sativa) containing a stably integrated dgt-28transgene is generated through Agrobacterium-mediated transformation ofsterilized rice seed. A disarmed Agrobacterium strain carrying a binaryvector containing a functional dgt-28 is used to initiatetransformation.

Culture media are adjusted to pH 5.8 with 1 M KOH and solidified with2.5 g/l Phytagel (Sigma-Aldrich, St. Louis, Mo.). Embryogenic calli arecultured in 100×20 mm petri dishes containing 30 ml semi-solid medium.Rice plantlets are grown on 50 ml medium in MAGENTA boxes. Cellsuspensions are maintained in 125 ml conical flasks containing 35 mLliquid medium and rotated at 125 rpm. Induction and maintenance ofembryogenic cultures occur in the dark at 25-26° C., and plantregeneration and whole-plant culture occur in illuminated room with a16-h photoperiod (Zhang et al. 1996).

Induction and maintenance of embryogenic callus is performed on amodified NB basal medium as described previously (Li et al. 1993),wherein the media is adapted to contain 500 mg/L glutamine. Suspensioncultures are initiated and maintained in SZ liquid medium (Zhang et al.1998) with the inclusion of 30 g/L sucrose in place of maltose. Osmoticmedium (NBO) consisting of NB medium with the addition of 0.256 M eachof mannitol and sorbitol. Herbicide resistant callus is selected on NBmedium supplemented with the appropriate herbicide selective agent for3-4 weeks. Pre-regeneration is performed on medium (PRH50) consisting ofNB medium with 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg/lα-naphthaleneacetic acid (NAA), 5 mg/l abscisic acid (ABA) and selectiveherbicide for 1 week. Regeneration of plantlets follow the culturing onregeneration medium (RNH50) comprising NB medium containing 2,4-D, 0.5mg/l NAA, and selective herbicide until putatively transgenic shoots areregenerated. Shoots are transferred to rooting medium with half-strengthMurashige and Skoog basal salts and Gamborg's B5 vitamins, supplementedwith 1% sucrose and selective herbicide.

Mature desiccated seeds of Oryza sativa L. japonica cv. Taipei 309 aresterilized as described in Zhang et al. 1996. Embryogenic tissues areinduced by culturing sterile mature rice seeds on NB medium in the dark.The primary callus approximately 1 mm in diameter, is removed from thescutellum and used to initiate cell suspension in SZ liquid medium.Suspensions are then maintained as described in Zhang 1996.Suspension-derived embryogenic tissues are removed from liquid culture3-5 days after the previous subculture and placed on NBO osmotic mediumto form a circle about 2.5 cm across in a petri dish and cultured for 4h prior to bombardment. Sixteen to twenty hours after bombardment,tissues are transferred from NBO medium onto NBH50 selection medium,ensuring that the bombarded surface is facing upward, and incubated inthe dark for 14-17 days. Newly formed callus is then separated from theoriginal bombarded explants and placed nearby on the same medium.Following an additional 8-12 days, relatively compact, opaque callus isvisually identified, and transferred to PRH50 pre-regeneration mediumfor 7 days in the dark. Growing callus, which become more compact andopaque is then subcultured onto RNH50 regeneration medium for a periodof 14-21 days under a 16-h photoperiod. Regenerating shoots aretransferred to MAGENTA boxes containing ½ MSH50 medium. Multiple plantsregenerated from a single explant are considered siblings and aretreated as one independent plant line. A plant is scored as positive forthe dgt-28 gene if it produces thick, white roots and grows vigorouslyon ½ MSH50 medium. Once plantlets reach the top of the MAGENTA boxes,they are transferred to soil in a 6-cm pot under 100% humidity for aweek, and then are moved to a growth chamber with a 14-h light period at30° C. and in the dark at 21° C. for 2-3 weeks before transplanting into13-cm pots in the greenhouse. Seeds are collected and dried at 37° C.for one week prior to storage at 4° C.

T₀ Analysis of Dgt-28 Rice.

Transplanted rice transformants obtained via an Agrobacteriumtransformation method were transplanted into media and acclimated togreenhouse conditions. All plants were sampled for PCR detection ofdgt-28 and results demonstrate twenty-two PCR positive events forpDAB110827 (TraP8::dgt-28) and a minimum of sixteen PCR positive eventsfor pDAB110828 (TraP23::dgt-28). Southern analysis for dgt-28 of the PCRpositive events demonstrated simple (1-2 copy) events for bothconstructs. Protein expression of selected T₀ events demonstrated DGT-28protein expression ranges from below levels of detection to 130 ng/cm².Selected T₀ events from construct pDAB110828 were treated with 2240 gae/ha DURANGO DMA™ as previously described and assessed 7 and 14 daysafter treatment. Data demonstrated robust tolerance to the rate ofglyphosate applied. All PCR positive plants were allowed to produced T₁seed for further characterization.

Dgt-28 Heritability in Rice.

A 100 plant progeny test was conducted on four T₁ lines of dgt-28 fromconstruct pDAB110827 containing the chloroplast transit peptide TraP8.The seeds were planted into pots filled with media. All plants were thensprayed with 560 g ae/ha DURANGO DMA™ for the selection of the dgt-28gene as previously described. After 7 DAT, resistant and sensitiveplants were counted. Two out of the four lines tested for each constructsegregated as a single locus, dominant Mendelian trait (3R:1S) asdetermined by Chi square analysis. Dgt-28 is a heritable glyphosateresistance gene in multiple species.

Postemergence Herbicide Tolerance in Dgt-28 Transformed T₁ Rice.

T₁ resistant plants from each event used in the progeny testing weregiven unique identifiers and sampled for zygosity analyses of the dgt-28gene. Zygosity data were used to assign 2 hemizygous and 2 homozygousreplicates to each rate of glyphosate applied allowing for a total of 4replicates per treatment. These plants were compared against wildtypekitaake rice. All plants were sprayed with a track sprayer set at 187L/ha. The plants were sprayed from a range of 560-2240 g ae/ha DURANGODMA™. All applications were formulated in water with the addition of 2%w/v ammonium sulfate (AMS). Plants were evaluated at 7 and 14 days aftertreatment. Plants were assigned an injury rating with respect to overallvisual stunting, chlorosis, and necrosis. The T₁ generation issegregating, so some variable response is expected due to difference inzygosity.

Spray results demonstrate at 7 DAT (days after treatment) minimalvegetative injury to elevated rates of glyphosate were detected (datanot shown).

TABLE 27 Visual injury data at 14 DAT demonstrates less than 15% meanvisual injury up to 2240 g ae/ha glyphosate. % Injury % Injury 20- Std.Range Application Rate <20% 40% >40% Ave Dev. (%) TraP8::dgt-28 Event 10 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 4 0 0 0.0 0.0 0 1120 gae/ha 4 0 0 0.0 0.0 0 2240 g ae/ha 4 0 0 0.0 0.0 0 TraP8::dgt-28 Event 20 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 4 0 0 3.8 4.8 0-10 1120g ae/ha 4 0 0 12.0 3.6 8-15 2240 g ae/ha 4 0 0 15.0 6.0 8-20Non-transformed control 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha0 0 4 81.3 2.5 80-85  1120 g ae/ha 0 0 4 95.0 5.8 90-100 2240 g ae/ha 00 4 96.3 4.8 90-100

Protein detection of DGT-28 was assessed for replicates from all four T₁lines tested from pDAB110827. Data demonstrated DGT-28 mean proteinranges from 20-82 ng/cm² and 21-209 ng/cm² for hemizygous and homozygousreplicates respectively. These results demonstrated stable proteinexpression to the T₁ generation and tolerance of dgt-28 rice up to 2240g ae/ha glyphosate following an application of 560 g ae/ha glyphosateused for selection.

Transformation of Tobacco with Dgt-28.

Tobacco (cv. Petit Havana) leaf pieces were transformed usingAgrobacterium tumefaciens containing the dgt-28 transgene. Singlecolonies containing the plasmid which contains the dgt-28 transgene wereinoculated into 4 mL of YEP medium containing spectinomycin (50 μg/mL)and streptomycin (125 μg/mL) and incubated overnight at 28° C. on ashaker at 190 rpm. The 4 mL seed culture was subsequently used toinoculate a 25 mL culture of the same medium in a 125 mL baffledErlenmeyer flask. This culture was incubated at 28° C. shaking at 190rpm until it reached an OD₆₀₀ of ˜1.2. Ten mL of Agrobacteriumsuspension were then placed into sterile 60×20 mm Petri™ dishes.

Freshly cut leaf pieces (0.5 cm²) from plants aseptically grown on MSmedium (Phytotechnology Labs, Shawnee Mission, Kans.,) with 30 g/Lsucrose in PhytaTrays™ (Sigma, St. Louis, Mo.) were soaked in 10 mL ofovernight culture of Agrobacterium for a few minutes, blotted dry onsterile filter paper and then placed onto the same medium with theaddition of 1 mg/L indoleacetic acid and 1 mg/L 6-benzylamino purine.Three days later, leaf pieces co-cultivated with Agrobacterium harboringthe dgt-28 transgene were transferred to the same medium with 5 mg/LBasta™ and 250 mg/L cephotaxime.

After 3 weeks, individual T₀ plantlets were transferred to MS mediumwith 10 mg/L Basta™ and 250 mg/L cephotaxime an additional 3 weeks priorto transplanting to soil and transfer to the greenhouse. Selected T₀plants (as identified using molecular analysis protocols describedabove) were allowed to self-pollinate and seed was collected fromcapsules when they were completely dried down. T₁ seedlings werescreened for zygosity and reporter gene expression (as described below)and selected plants containing the dgt-28 transgene were identified.

Plants were moved into the greenhouse by washing the agar from theroots, transplanting into soil in 13.75 cm square pots, placing the potinto a Ziploc® bag (SC Johnson & Son, Inc.), placing tap water into thebottom of the bag, and placing in indirect light in a 30° C. greenhousefor one week. After 3-7 days, the bag was opened; the plants werefertilized and allowed to grow in the open bag until the plants weregreenhouse-acclimated, at which time the bag was removed. Plants weregrown under ordinary warm greenhouse conditions (27° C. day, 24° C.night, 16 hour day, minimum natural+supplemental light=1200 μE/m²s¹).

Prior to propagation, T₀ plants were sampled for DNA analysis todetermine the insert dgt-28 copy number by real-time PCR. Fresh tissuewas placed into tubes and lyophilized at 4° C. for 2 days. After thetissue was fully dried, a tungsten bead (Valenite) was placed in thetube and the samples were subjected to 1 minute of dry grinding using aKelco bead mill. The standard DNeasy™ DNA isolation procedure was thenfollowed (Qiagen, DNeasy 69109). An aliquot of the extracted DNA wasthen stained with Pico Green (Molecular Probes P7589) and read in thefluorometer (BioTek™) with known standards to obtain the concentrationin ng/μl. A total of 100 ng of total DNA was used as template. The PCRreaction was carried out in the 9700 Geneamp™ thermocycler (AppliedBiosystems), by subjecting the samples to 94° C. for 3 minutes and 35cycles of 94° C. for 30 seconds, 64° C. for 30 seconds, and 72° C. for 1minute and 45 seconds followed by 72° C. for 10 minutes. PCR productswere analyzed by electrophoresis on a 1% agarose gel stained with EtBrand confirmed by Southern blots.

Five to nine PCR positive events with 1-3 copies of dgt-28 gene from 3constructs containing a different chloroplast transit peptide sequence(TraP4, TraP8 and TraP23) were regenerated and moved to the greenhouse.

All PCR positive plants were sampled for quantification of the DGT-28protein by standard ELISA. DGT-28 protein was detected in all PCRpositive plants and a trend for an increase in protein concentration wasnoted with increasing copy number of dgt-28.

Aad-12 (v1) Heritability in Tobacco.

A 100 plant progeny test was conducted on five T₁ lines of dgt-28 perconstruct. Constructs contained one of the following chloroplast transitpeptide sequences: TraP4, TraP8 or TraP23. The seeds were stratified,sown, and transplanted with respect much like that of the Arabidopsisprocedure exemplified above, with the exception that null plants werenot removed by in initial selection prior to transplanting. All plantswere then sprayed with 280 g ae/ha IGNITE 280 SL for the selection ofthe pat selectable marker as previously described. After 3 DAT,resistant and sensitive plants were counted.

Four out of the five lines tested for each construct segregated as asingle locus, dominant Mendelian trait (3R:1S) as determined by Chisquare analysis. Dgt-28 is a heritable glyphosate resistance gene inmultiple species.

Postemergence Herbicide Tolerance in Dgt-28 Transformed T₁ Tobacco.

T₁ resistant plants from each event used in the progeny testing weregiven unique identifiers and sampled for zygosity analyses of the dgt-28gene. Zygosity data were used to assign 2 hemizygous and 2 homozygousreplicates to each rate of glyphosate applied allowing for a total of 4replicates per treatment. These plants were compared against wildtypePetite havana tobacco. All plants were sprayed with a track sprayer setat 187 L/ha. The plants were sprayed from a range of 560-4480 g ae/haDURANGO DMA™. All applications were formulated in water with theaddition of 2% w/v ammonium sulfate (AMS). Plants were evaluated at 7and 14 days after treatment. Plants were assigned an injury rating withrespect to overall visual stunting, chlorosis, and necrosis. The T₁generation is segregating, so some variable response is expected due todifference in zygosity.

Spray results demonstrate at 7 DAT (days after treatment) minimalvegetative injury to elevated rates of glyphosate were detected (datanot shown). Following 14 DAT, visual injury data demonstrates increasedinjury with single copy events of the construct containing TraP4compared to single copy events from the constructs TraP8 and TraP23.Table 28.

TABLE 28 At a rate of 2240 g ae/ha glyphosate, an average injury of37.5% was demonstrated with the event containing TraP4, where eventscontaining TraP8 and TraP23 demonstrated an average injury of 9.3% and9.5% respectively. % Injury % Injury 20- Std. Range Application Rate<20% 40% >40% Ave Dev. (%) TraP4::dgt-28 (pDAB107543) 0 g ae/haglyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 2 2 0 18.0 8.1 10-25 1120 g ae/ha1 3 0 24.5 4.9 18-30 2240 g ae/ha 0 3 1 37.5 6.5 30-45 4480 g ae/ha 0 22 42.5 2.9 40-45 TraP8::dgt-28 (pDAB107545) 0 g ae/ha glyphosate 4 0 00.0 0.0 0 560 g ae/ha 4 0 0 3.3 3.9 0-8 1120 g ae/ha 4 0 0 6.5 1.7 5-82240 g ae/ha 4 0 0 9.3 3.0  5-12 4480 g ae/ha 2 2 0 17.5 6.5 10-25TraP23::dgt-28 (pDAB107553) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 gae/ha 4 0 0 10.0 1.6  8-12 1120 g ae/ha 4 0 0 8.8 3.0  5-12 2240 g ae/ha4 0 0 9.5 4.2  5-15 4480 g ae/ha 4 0 0 15.8 1.5 15-18 Petite havana 0 gae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 0 0 4 85.0 4.1 80-90 1120 gae/ha 0 0 4 91.3 2.5 90-95 2240 g ae/ha 0 0 4 94.5 3.3 90-98 4480 gae/ha 0 0 4 98.3 2.4  95-100

These results demonstrated tolerance of dgt-28 up to 4480 g ae/haglyphosate, as well as differences in tolerance provided by chloroplasttransit peptide sequences linked to the dgt-28 gene.

Dgt-28 Protection Against Elevated Glyphosate Rates in the T₂Generation.

A 25 plant progeny test was conducted on two to three T₂ lines of dgt-28per construct. Homozygous lines were chosen based on zygosity analysescompleted in the previous generation. The seeds were stratified, sown,and transplanted as previously described. All plants were then sprayedwith 280 g ae/ha Ignite 280 SL for the selection of the pat selectablemarker as previously described. After 3 DAT, resistant and sensitiveplants were counted. All lines tested for each construct did notsegregate thereby confirming homogeneous lines in the T₂ generation anddemonstrating Mendelian inheritance through at least two generation ofdgt-28 in tobacco.

Rates of DURANGO DMA™ ranging from 420-3360 g ae/ha glyphosate wereapplied to 2-3 leaf tobacco as previously described. Visual injury data14 DAT confirmed the tolerance results that were demonstrated in the T₁generation. Foliar results from a two copy lines from the constructcontaining TraP4 demonstrated similar tolerance to that of single copyTraP8 and TraP23 lines (data not shown).

TABLE 29 Single copy lines from the construct containing TraP4 withdgt-28 demonstrated increased injury compared to lines from constructscontaining TraP8 and TraP23 with dgt-28. % Injury % Injury 20- Std.Range Application Rate <20% 40% >40% Ave Dev. (%) TraP4::dgt-28(pDAB107543) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g ae/ha 0 4 0 23.84.8 20-30 840 g ae/ha 0 4 0 30.0 4.1 25-35 1680 g ae/ha 0 4 0 35.0 5.830-40 3360 g ae/ha 0 4 0 31.3 2.5 30-35 TraP8::dgt-28 (pDAB107545) 0 gae/ha glyphosate 4 0 0 0.0 0.0 0 420 g ae/ha 4 0 0 0.0 0.0 0 840 g ae/ha4 0 0 2.5 2.9 0-5 1680 g ae/ha 4 0 0 9.3 3.4  5-12 3360 g ae/ha 4 0 010.5 1.0 10-12 TraP23::dgt-28 (pDAB107553) 0 g ae/ha glyphosate 4 0 00.0 0.0 0 420 g ae/ha 4 0 0 0.0 0.0 0 840 g ae/ha 4 0 0 6.3 2.5  5-101680 g ae/ha 4 0 0 10.0 0.0 10  3360 g ae/ha 3 1 0 13.8 4.8 10-20 Petitehavana 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g ae/ha 0 0 4 95.0 0.095  840 g ae/ha 0 0 4 98.8 1.0  98-100 1680 g ae/ha 0 0 4 99.5 1.0 98-100 3360 g ae/ha 0 0 4 100 0.0 100 

The data demonstrate robust tolerance of dgt-28 tobacco up to 3360 gae/ha glyphosate through two generations compared to the non-transformedcontrol.

Selected plants from each event were sampled prior to glyphosateapplications for analyses of the DGT-28 protein by standard DGT-28ELISA. Data demonstrated DGT-28 mean protein expression of the simple(1-2 copy) lines across constructs ranging from 72.8-114.5 ng/cm². Datademonstrates dgt-28 is expressing protein in the T₂ generation oftransformed tobacco and tolerance data confirms functional DGT-28protein.

Stacking of Dgt-28 to Increase Herbicide Spectrum in Tobacco (Cv. PetitHavana).

Homozygous dgt-28 (pDAB107543 and pDAB107545) and aad-12 v1 (pDAB3278)plants (see PCT/US2006/042133 for the latter, which is incorporatedherein by this reference in its entirety) were both reciprocally crossedand F₁ seed was collected. The F₁ seed from two reciprocal crosses ofeach gene were stratified and treated 6 reps of each cross were treatedwith 1120 g ae/ha glyphosate (selective for the dgt-28 gene), 1120 gae/ha 2,4-D (selective for the aad-12 gene), or a tank mixture of thetwo herbicides at the rates described. Plants were graded at 14 DAT.Spray results are shown in Table 30.

TABLE 30 Response of F₁ aad-12 and dgt-28 aad-12 × aad-12 × PetiteTraP4::dgt-28 TraP8::dgt-28 havana Application Rate Tolerance 1120 gae/ha 2,4-D ++++ ++++ − 1120 g ae/ha glyphosate ++ ++ − 1120 g ae/ha2,4-D + ++ ++ − 1120 g ae/ha glyphosate

The results confirm that dgt-28 can be successfully stacked with aad-12(v1), thus increasing the spectrum herbicides that may be applied to thecrop of interest (glyphosate+phenoxyactetic acids for dgt-28 and aad-12,respectively). In crop production where hard to control broadleaf weedsor resistant weed biotypes exist the stack can be used as a means ofweed control and protection of the crop of interest. Additional input oroutput traits could also be stacked with the dgt-28 gene.

Resistance to Glyphosate in Wheat. Production of Binary Vectors EncodingDGT-28.

Binary vectors containing DGT-28 expression and PAT selection cassetteswere designed and assembled using skills and techniques commonly knownin the art. Each DGT-28 expression cassette contained the promoter, 5′untranslated region and intron from the Ubiquitin (Ubi) gene from Zeamays (Toki et al., Plant Physiology 1992, 100 1503-07), followed by acoding sequence consisting of one of four transit peptides (TraP4,TraP8, TraP23 or TraP5) fused to the 5′ end of a synthetic version ofthe 5-enolpyruvylshikimate-3-phosphate synthase gene (DGT-28), which hadbeen codon optimized for expression in plants. The DGT-28 expressioncassette terminated with a 3′ untranslated region (UTR) comprising thetranscriptional terminator and polyadenylation site of a lipase gene(Vpl) from Z. mays (Paek et al., Mol. Cells 1998 30; 8(3) 336-42). ThePAT selection cassette comprised of the promoter, 5′ untranslated regionand intron from the Actin (Act1) gene from Oryza sativa (McElroy et al.,The Plant Cell 1990 2(2) 163-171), followed by a synthetic version ofthe phosphinothricin acetyl transferase (PAT) gene isolated fromStreptomyces viridochromogenes, which had been codon optimized forexpression in plants. The PAT gene encodes a protein that confersresistance to inhibitors of glutamine synthetase comprisingphophinothricin, glufosinate, and bialaphos (Wohlleben et al., Gene1988, 70(1), 25-37). The selection cassette was terminated with the 3′UTR comprising the transcriptional terminator and polyadenylation sitesfrom the 35s gene of cauliflower mosaic virus (CaMV) (Chenault et al.,Plant Physiology 1993 101 (4), 1395-1396).

The selection cassette was synthesized by a commercial gene synthesisvendor (GeneArt, Life Technologies) and cloned into a Gateway-enabledbinary vector. The DGT-28 expression cassettes were sub-cloned intopDONR221. The resulting ENTRY clone was used in a LR Clonase II(Invitrogen, Life Technologies) reaction with the Gateway-enabled binaryvector encoding the phosphinothricin acetyl transferase (PAT) expressioncassette. Colonies of all assembled plasmids were initially screened byrestriction digestion of purified DNA using restriction endonucleasesobtained from New England BioLabs (NEB; Ipswich, Mass.) and Promega(Promega Corporation, WI). Plasmid DNA preparations were performed usingthe QlAprep Spin Miniprep Kit (Qiagen, Hilden) or the Pure Yield PlasmidMaxiprep System (Promega Corporation, WI), following the instructions ofthe suppliers. Plasmid DNA of selected clones was sequenced using ABISanger Sequencing and Big Dye Terminator v3.1 cycle sequencing protocol(Applied Biosystems, Life Technologies). Sequence data were assembledand analyzed using the SEQUENCHER™ software (Gene Codes Corporation, AnnArbor, Mich.).

The resulting four binary expression clones: pDAS000122 (TraP4-DGT28),pDAS000123 (TraP8-DGT28), pDAS000124 (TraP23-DGT28) and pDAS000125(TraP5-DGT28) were each transformed into Agrobacterium tumefaciensstrain EHA105.

Production of Transgenic Wheat Events with Dgt-28 Expression Construct.

Transgenic wheat plants expressing one of the four DGT-28 expressionconstructs were generated by Agrobacterium-mediated transformation usingthe donor wheat line Bobwhite MPB26RH, following a protocol similar toWu et al., Transgenic Research 2008, 17:425-436. Putative T0 transgenicevents were selected for phosphinothricin (PPT) tolerance, the phenotypeconferred by the PAT selectable marker, and transferred to soil. The T0plants were grown under glasshouse containment conditions and T₁ seedwas produced. Overall, about 45 independent T0 events were generated foreach DGT-28 expression construct.

Glyphosate Resistance in T₀ Wheat Dgt-28 Wheat Events.

T₀ events were allowed to acclimate in the greenhouse and were grownuntil 2-4 new, normal looking leaves had emerged from the whorl (i.e.,plants had transitioned from tissue culture to greenhouse growingconditions). Plants were grown at 25° C. under 12 hour of supplementallighting in the greenhouse until maturity. An initial screen ofglyphosate tolerance and Taqman analyses was completed on T₁ plantsgrown under the same conditions as previously described. Data allowedfor determination of heritable T₁ events to be further characterized.Six low copy (1-2 copy) and two multi-copy T₁ events were replantedunder greenhouse conditions and grown until the 3 leaf stage. T₁ plantswere sprayed with a commercial formulation of glyphosate (Durango DMA′)from a range of 420-3360 g ae/ha, which are capable of significantinjury to untransformed wheat lines. The addition of 2% w/v ammoniumsulfate was included in the application. A lethal dose is defined as therate that causes >75% injury to the Bob White MPB26RH non-transformedcontrol. Herbicide was applied.

In this example, the glyphosate applications were utilized for bothdetermining the segregation of the dgt-28 gene in the T₁ generation aswell as demonstrating tolerance to increasing levels of glyphosate. Theresponse of the plants is presented in terms of a scale of visual injury21 days after treatment (DAT). Data are presented as a histogram ofindividuals exhibiting less than 25% visual injury (4), 25%-50% visualinjury (3), 50%-75% visual injury (2) and greater than 75% injury (1).An arithmetic mean and standard deviation is presented for eachconstruct used for wheat transformation. The scoring range of individualresponse is also indicated in the last column for each rate andtransformation. Wild-type, non-transformed wheat (c.v. Bob WhiteMPB26RH) served as a glyphosate sensitive control. In the T₁ generationhemizygous and homozygous plants were available for testing for eachevent and therefore were included for each rate of glyphosate tested.Hemizygous plants will contain half of the dose of the gene ashomozygous plants, therefore variability of response to glyphosate maybe expected in the T₁ generation.

The results of the T₁ dgt-28 wheat plants demonstrated that tolerance toglyphosate was achieved at rates up to 3360 g ae/ha with the chloroplasttransit peptides TraP4, TraP5, TraP8 and TraP23. Table 31. Data are of alow copy T₁ event but are representative of the population for eachconstruct.

TABLE 31 Response of low copy T₁ dgt-28 wheat events to glyphosate 21days after treatment. % Injury % Injury 25- 50- Std. Range ApplicationRate <25% 50% 75% >75% Ave Dev. (%) TraP4::dgt-28 420 g ae/ha 5 0 0 04.00 0.00 4 840 g ae/ha 6 2 0 0 3.75 0.46 3-4 1680 g ae/ha 4 2 0 0 3.670.52 3-4 3360 g ae/ha 4 2 0 0 3.67 0.52 3-4 TraP8::dgt-28 420 g ae/ha 53 0 0 3.63 0.52 3-4 840 g ae/ha 3 5 0 0 3.38 0.52 3-4 1680 g ae/ha 4 3 00 3.57 0.53 3-4 3360 g ae/ha 5 5 0 0 3.50 0.53 3-4 TraP23::dgt-28 420 gae/ha 9 2 0 0 3.82 0.40 3-4 840 g ae/ha 8 1 0 0 3.89 0.33 3-4 1680 gae/ha 7 5 0 0 3.58 0.0 3-4 3360 g ae/ha 8 2 0 0 3.80 4.8 3-4TraP5::dgt-28 420 g ae/ha 5 2 0 0 3.71 0.49 3-4 840 g ae/ha 4 2 0 0 3.670.52 3-4 1680 g ae/ha 7 3 0 0 3.70 0.48 3-4 3360 g ae/ha 6 0 0 0 4.000.00 3-4 Bobwhite MPB26RH 420 g ae/ha 0 1 1 10 1.25 0.62 1-3 840 g ae/ha0 0 0 10 1.00 0.00 1 1680 g ae/ha 0 0 0 12 1.17 0.58 1-3 3360 g ae/ha 00 0 10 1.00 0.00 1

At 21 DAT, resistant and sensitive plants are counted to determine thepercentage of lines that segregated as a single locus, dominantMendelian trait (3R:1S) as determined by Chi square analysis. Table 32.These data demonstrate that dgt-28 is inheritable as a robust glyphosateresistance gene in a monocot species.

TABLE 32 Percentage of T₁ dgt-28 events by construct that demonstratedheritablity in a mendelian fashion based off of a glyphosate selectionat rates ranging from 420-3360 g ae/ha. % T₁ events % T₁ events testedthat tested that No. T₁ Construct segregated at a segregated as eventsID CTP:GOI single locus 2 loci tested pDAS000122 TraP4::dgt-28 62.5%37.5% 8 pDAS000123 TraP8::dgt-28 87.5% 12.5% 8 pDAS000124 TraP23::dgt-2812.5% 87.5% 8 pDAS000125 TraP5::dgt-28 62.5% 0.0% 8

Example 4: Chimeric Chloroplast Transit Peptide (TraP) Sequences forExpression of Agronomically Important Transgenes in Maize Cry2Aa:

The Cry2Aa protein from Bacillus thuringiensis has demonstrated activityagainst Helicoverpa zea (CEW) and Ostrinia nubilalis (ECB). A singleversion of the cry2Aa gene (SEQ ID NO:10), codon biased for maize, wastested in maize. In this experiment, Cry2Aa was evaluated alone and inconjunction with the TraP8 chimeric chloroplast transit peptide in maizeto determine the insect tolerance activity and to evaluate the effectthe TraP8 v2 chimeric chloroplast transit peptide sequence would have onthe expression of the Cry2Aa protein in maize.

The pDAB109807 construct which contains the Trap8 v2 chimericchloroplast transit peptide sequence (SEQ ID NO:8) and a GCA codonlinker were cloned upstream of the cry2Aa gene and incorporated intoconstruct pDAB109807 (FIG. 12) for insect tolerance testing in maizeplants. The resulting constructs contained two plant transcription units(PTU). The first PTU comprised the Zea mays Ubiquitin 1 promoter (ZmUbi1promoter; Christensen, A., Sharrock R., and Quail P., (1992) Maizepolyubiquitin genes: structure, thermal perturbation of expression andtranscript splicing, and promoter activity following transfer toprotoplasts by electroporation, Plant Molecular Biology, 18:675-689),TraP8-cry2Aa fusion gene (TraP8 Cry2Aa), and Zea mays Lipase 3′untranslated region (ZmLip 3′UTR; U.S. Pat. No. 7,179,902). Theconstructs were confirmed via restriction enzyme digestion andsequencing. The second PTU comprised the Sugar Cane Bacilliform Viruspromoter (SCBV promoter; U.S. Pat. No. 6,489,462), aad-1 herbicidetolerance gene containing a MSV leader and alcohol dehydrogenase 1intron 6 (AAD-1; U.S. Pat. No. 7,838,733, and MSV Leader sequence;Genbank Acc. No. FJ882146.1, and the alcohol dehydrogenase intron;Genbank Acc. No. EF539368.1), and Zea mays Lipase 3′ untranslated region(ZmLip 3′UTR). A control plasmid, pDAB107687, which did not contain achloroplast transit peptide sequence upstream of the cry2Aa gene wasbuilt and included in the studies (FIG. 13). The plasmids wereintroduced into Agrobacterium tumefaciens for plant transformation.

Ears from Zea mays cultivar B104 were harvested 10-12 days postpollination. Harvested ears were de-husked and surface-sterilized byimmersion in a 20% solution of commercial bleach (Ultra Clorox®Germicidal Bleach, 6.15% sodium hypochlorite) and two drops of Tween 20,for 20 minutes, followed by three rinses in sterile, deionized waterinside a laminar flow hood. Immature zygotic embryos (1.8-2.2 mm long)were aseptically excised from each ear and distributed into one or moremicro-centrifuge tubes containing 2.0 ml of Agrobacterium suspensioninto which 2 μl of 10% Break-Thru® 5233 surfactant had been added.

Upon completion of the embryo isolation activity the tube of embryos wasclosed and placed on a rocker platform for 5 minutes. The contents ofthe tube were then poured out onto a plate of co-cultivation medium andthe liquid Agrobacterium suspension was removed with a sterile,disposable, transfer pipette. The co-cultivation plate containingembryos was placed at the back of the laminar flow hood with the lidajar for 30 minutes; after which time the embryos were oriented with thescutellum facing up using a microscope. The co-cultivation plate withembryos was then returned to the back of the laminar flow hood with thelid ajar for a further 15 minutes. The plate was then closed, sealedwith 3M Micropore tape, and placed in an incubator at 25° C. with 24hours/day light at approximately 60 μmol m-2 s-1 light intensity.

Following the co-cultivation period, embryos were transferred to Restingmedium. No more than 36 embryos were moved to each plate. The plateswere wrapped with 3M micropore tape and incubated at 27° C. with 24hours/day light at approximately 50 μmol m-2 s-1 light intensity for7-10 days. Callused embryos were then transferred onto Selection Imedium. No more than 18 callused embryos were moved to each plate ofSelection I. The plates were wrapped with 3M micropore tape andincubated at 27° C. with 24 hours/day light at approximately 50 μmol m-2s-1 light intensity for 7 days. Callused embryos were then transferredto Selection II medium. No more than 12 callused embryos were moved toeach plate of Selection II. The plates were wrapped with 3M microporetape and incubated at 27° C. with 24 hours/day light at approximately 50μmol m-2 s-1 light intensity for 14 days.

At this stage resistant calli were moved to Pre-Regeneration medium. Nomore than 9 calli were moved to each plate of Pre-Regeneration. Theplates were wrapped with 3M micropore tape and incubated at 27° C. with24 hours/day light at approximately 50 μmol m-2 s-1 light intensity for7 days. Regenerating calli were then transferred to Regeneration mediumin Phytatrays™ and incubated at 28° C. with 16 hours light/8 hours darkper day at approximately 150 μmol m-2 s-1 light intensity for 7-14 daysor until shoots develop. No more than 5 calli were placed in eachPhytatray™. Small shoots with primary roots were then isolated andtransferred to Shoot/Root medium. Rooted plantlets about 6 cm or tallerwere transplanted into soil and moved out to a growth chamber forhardening off.

Transgenic plants were assigned unique identifiers through andtransferred on a regular basis to the greenhouse. Plants weretransplanted from Phytatrays™ to small pots (T. O. Plastics, 3.5-inchSVD, 700022C) filled with growing media (Premier Tech Horticulture,ProMix BX, 0581 P) and covered with humidomes to help acclimate theplants. Plants were placed in a Conviron™ growth chamber (28° C./24° C.,16-hour photoperiod, 50-70% RH, 200 μmol light intensity) until reachingV3-V4 stage. This aided in acclimating the plants to soil and harshertemperatures. Plants were then moved to the greenhouse (Light ExposureType: Photo or Assimilation; High Light Limit: 1200 PAR; 16-hour daylength; 27° C. Day/24° C. Night) and transplanted from the small pots to5.5 inch pots. Approximately 1-2 weeks after transplanting to largerpots plants were sampled for bioassay. One plant per event wasbioassayed.

Select events were identified for advancement to the next generationbased on copy number of the genes, protein detection by Western blot andactivity against the bioassay insects. Events that contained theSpectinomycin resistance gene were noted but not necessarily omittedfrom advancement. Events selected for advancement were transplanted into5 gallon pots. Observations were taken periodically to track anyabnormal phenotypes. Shoot bags were placed over the shoots prior tosilk emergence to prevent cross-contamination by stray pollen. Anyshoots producing silks prior to covering were noted and the shoot wasremoved. The second shoot was then covered and used for pollinations.Plants that produced abnormal or no shoots were recorded in thedatabase. Silks were cut back the day prior to pollinations to providean even brush to accept pollen and the plants were self pollinated.

Plants for T1 selection were sprayed at 7 days post sowing. They weregrown in 4-inch pots of Metro 360 potting soil with 15 pots per flat.Seedling growth stage was V1-V1.5. Pots with poor germination or containvery small plants (whorl still closed) were marked so they were notincluded in the selection assessment. Whole flats of plants were thenplaced in secondary carrier trays for track sprayer application. Trayswere placed two at a time in the Mandel track sprayer, calibrated todeliver a volume 187 L/ha to the target area using an 8002E flat fannozzle (Tee Jet). A solution of 35 g ae/ha Assure II (quizalofop)+1% COC(crop oil concentrate) was formulated for the application. A volume of15 mls./spray was used to calculate the total spray solution needed.Calculations; (35 g ae/ha)×(1 ha/187L)×(1 L/97.7 g ae Assure II)=0.192%solution or 28.74 μl/15 ml H2O+1% v/v). After application, the plantswere then allowed to dry for one hour in spray lab before returning togreenhouse. Approximately 1-2 weeks after transplanting to larger potsplants were sampled for bioassay. One plant per event was bioassayed.

All of the T₀ events that passed the molecular analysis screen wereanalyzed for Cry2Aa protein expression levels. The events from thecontrol construct, pDAB107687, which comprised Cry2Aa without a TraP hadsignificantly higher average expression level of Cry2Aa (15.0 ng/cm2) ascompared to events from pDAB109807 (5.0 ng/cm2) which contained TraP8.Despite the reduced levels of expression of the pDAB109807 events, theseevents still expressed the Cry2Aa protein.

The T1 events were also analyzed were analyzed for Cry2Aa proteinexpression levels. The events from the control construct, pDAB107687,which comprised Cry2Aa without a TraP had significantly higher averageexpression level of Cry2Aa (55 and 60 ng/cm²) as compared to events frompDAB109807 (about 20 to 40 ng/cm²) which contained TraP8. Despite thereduced levels of expression of the pDAB109807 events, these eventsstill expressed the Cry2Aa protein.

Transgenic plants containing single Bt genes were tested forinsecticidal activity in bioassays conducted with neonate lepidopteranlarvae on leaves from the transgenic plants. The lepidopteran speciesassayed were the European Corn Borer, Ostrinia nubilalis (Hubner) (ECB),and the Corn Earworm, Helicoverpa zea (CEW).

32-well trays (C-D International, Pitman, N.J.) were partially filledwith a 2% agar solution and agar was allowed to solidify. Leaf sectionsapproximately 1 in² were taken from each plant and placed singly intowells of the 32-well trays. One leaf piece was placed into each well,and two leaf pieces were tested per plant and per insect. Insects weremass-infested using a paintbrush, placing 10 neonate larvae into eachwell. Trays were sealed with perforated sticky lids which allowedventilation during the test. Trays were placed at 28° C., 40% RH, 16hours light: 8 hours dark for three days. After the duration of thetest, a simple percent damage score was taken for each leaf piece.Damage scores for each test were averaged and used alongside proteinexpression analysis to conduct correlation analyses.

The results of the T₀ and T₁ bioassay indicated that the TraP8 chimericchloroplast transit peptide sequence was functional and that thepDAB109807 events provided protection against the tested insects. In theT1 events, the plants expressing the Cry2Aa protein without a TraP(pDAB107687) had a mean leaf damage that was not significantly differentthan the plant expressing the Cry2Aa protein with TraP8 (pDAB109807)across all insect species tested. These results were surprising, giventhat the plants expressing the Cry2Aa protein without a TraP(pDAB107687) expressed higher levels of protein as compared to theplants expressing the Cry2Aa protein with TraP8 (pDAB109807).

VIP3Ab1:

The Vip3Ab1 protein from Bacillus thuringiensis has demonstratedactivity against Helicoverpa zea (CEW) and Fall Armyworm (FAW) andresistant Fall Armyworm (rFAW). The vip3Ab1 v6 (SEQ ID NO:11) andvip3Ab1 v7 (SEQ ID NO:12) genes were expressed and tested for insecttolerance in maize. In this experiment, vip3Ab1v6 and vip3Ab1 v7 wereevaluated alone and in conjunction with the TraP8 chimeric chloroplasttransit peptide in maize to determine the insect tolerance activity andto evaluate the effect the TraP8 v2 chimeric chloroplast transit peptidesequence would have on the expression of the Vip3Ab1 v6 and Vip3Ab1 v7proteins in maize.

The pDAB111481 (FIG. 14) construct which contains the Trap8 v2 chimericchloroplast transit peptide-encoding polynucleotide sequence (SEQ IDNO:8) and a GCA codon linker were cloned upstream of the vip3ab1 v6 geneand tested for insect tolerance in maize plants. The resulting constructcontained two plant transcription units (PTU). The first PTU comprisedthe Zea mays Ubiquitin 1 promoter (ZmUbi1 promoter; Christensen, A.,Sharrock R., and Quail P., (1992) Maize polyubiquitin genes: structure,thermal perturbation of expression and transcript splicing, and promoteractivity following transfer to protoplasts by electroporation, PlantMolecular Biology, 18:675-689), TraP8-vip3ab1 v6 fusion gene(TraP8-Vip3Ab1v6) and Zea mays Peroxidase5 3′ untranslated region (ZmPer5 3′UTR). The construct was confirmed via restriction enzyme digestionand sequencing. The second PTU comprised the Sugar Cane BacilliformVirus promoter (SCBV promoter; U.S. Pat. No. 6,489,462), aad-1 herbicidetolerance gene containing a MSV leader and alcohol dehydrogenase 1intron 6 (AAD-1; U.S. Pat. No. 7,838,733, and MSV Leader sequence;Genbank Acc. No. FJ882146.1, and the alcohol dehydrogenase intron;Genbank Acc. No. EF539368.1), and Zea mays Lipase 3′ untranslated region(ZmLip 3′UTR). A control plasmid, pDAB111479, which did not contain achloroplast transit peptide sequence upstream of the vip3ab1 v6 gene wasbuilt and included in the studies (FIG. 15). The plasmids wereintroduced into Agrobacterium tumefaciens for plant transformation.

The pDAB111338 (FIG. 16) construct which contains the Trap8 v2 chimericchloroplast transit peptide sequence (SEQ ID NO:8) and a GCA codonlinker were cloned upstream of the vip3ab1 v7 gene and tested for insecttolerance testing in maize plants. The resulting construct contained twoplant transcription units (PTU). The first PTU was comprised of the Zeamays Ubiquitin 1 promoter (ZmUbi1 promoter; Christensen, A., SharrockR., and Quail P., (1992) Maize polyubiquitin genes: structure, thermalperturbation of expression and transcript splicing, and promoteractivity following transfer to protoplasts by electroporation, PlantMolecular Biology, 18:675-689), TraP8-Vip3Ab1v7 fusion gene(TraP8-vip3ab1 v7) and Zea mays Peroxidase5 3′ untranslated region(ZmPer 5 3′UTR). The construct was confirmed via restriction enzymedigestion and sequencing. The second PTU was comprised of the Sugar CaneBacilliform Virus promoter (SCBV promoter; U.S. Pat. No. 6,489,462),aad-1 herbicide tolerance gene containing a MSV leader and alcoholdehydrogenase 1 intron 6 (AAD-1; U.S. Pat. No. 7,838,733, and MSV Leadersequence; Genbank Acc. No. FJ882146.1, and the alcohol dehydrogenaseintron; Genbank Acc. No. EF539368.1), and Zea mays Lipase 3′untranslated region (ZmLip 3′UTR). A control plasmid, pDAB112710, whichdid not contain a chloroplast transit peptide sequence upstream of theVip3Ab1v7 gene was built and included in the studies (FIG. 17). Theplasmids were cloned into Agrobacterium tumefaciens for planttransformation.

Maize transformation, protein expression and insect bioassays werecompleted following the protocols previously described, and the resultsare shown in Table 33. The results of insect bioassays indicated thatthe TraP8 chimeric chloroplast transit peptide sequence was functionaland that the pDAB111338 and pDAB111481 events provided protectionagainst the insects tested in bioassay. In the tested events, the plantsexpressing the Vip3Ab1 protein without a TraP, (pDAB112710 andpDAB111479), had a mean leaf damage that was not significantly differentthan the plant expressing the Vip3Ab1 protein with TraP8 (pDAB111338 andpDAB111481). In conclusion, the Western blots and bioassays indicatedthat all of the tested events expressed the Vip3 Ab1 protein.

TABLE 33 Results of the biochemical and bioassay results for Vip3Ab1 v6and Vip3Ab1 v7 coding sequences that were fused to TraP8 as compared toVip3Ab1 v6 and Vip3Ab1 v7 coding sequences that did not possess achloroplast transit peptide sequence. Biochemical Assay Results BioAssayResults Method of analysis CEW FAW rFAW ELISA (ng/cm₂) Western TotalMean Total Mean Total Mean Average LC/MS/MS positive Events CEW % LeafFAW % Leaf rFAW % Leaf Plasmid Description expression (fmole/cm₂) eventstested damage Damage damage Damage damage Damage pDAB111479 Vip3Ab1 v659 ELISA 14/17 19 205.0 10.8 368.0 19.4 325.0 17.1 No TraP pDAB111481Vip3Ab1 v6 239 ELISA 4/4 17 124.0 7.3 110.0 6.5 77.0 4.5 Trap8 v2pDAB112710 Vip3Ab1 v7 143 ELISA 18/20 20 79.0 4.0 107.0 5.4 117.0 5.9 NoTraP pDAB111338 Vip3Ab1 v7 180 ELISA 5/6 9 63.0 7.0 99.0 11.0 111.0 12.3Trap8 v2

Example 5: In Planta Cleavage of Chimeric Chloroplast Transit Peptide(TraP) Sequences

The cleavage site of the TraP8 and TraP9 chimeric chloroplast transitpeptide was determined via MALDI spectrometry and N-terminal Edmandegradation sequencing. Plant material was obtained from transgenicplants which contained the TraP8-dgt/4, TraP8-dgt28, TraP9-dgt/4, andTraP9-dgt28 fusion genes and assayed to determine the location ofcleavage of the chimeric chloroplast transit peptide occurred duringtranslocation within the chloroplast.

MALDI Results:

The semi-purified proteins from a plant sample were separated bySDS-PAGE. The bands of protein of a size equivalent to the molecularweight of YFP were excised from the gel, de-stained and dried. Next, thedried protein bands were in-gel digested with Trypsin (Promega; Madison,Wis.) in 25 mM ammonium bicarbonate for overnight at 37° C. The peptideswere purified by a C18 ZipTip™ (Millipore, Bedford, Mass.) and elutedwith 50% acetonitrile/0.1% TFA. The samples were mixed with matrixα-cyano-4-hydroxycinnamic acid in a 1:1 ratio and the mix was sportedonto a MALDI sample plate and air dried.

The peptide mass spectrum was generated using a Voyager DE-PRO MALDI-TOFMass Spectrometer™ (Applied Biosystems; Framingham, Mass.). Externalcalibration was performed by using a Calibration Mixture 2™ (AppliedBiosystems). Internal calibration was performed using the trypsinautolysis peaks at m/z 842.508, 1045.564 and 2211.108. All mass spectrawere collected in the positive ion reflector model. The peptide massfingerprint (PMF) analysis was conducted using PAWS™ (Protein AnalysisWorkSheet) freeware from Proteometrics LLC by matching the PMF of thesample with theoretical PMF of target protein to verify if the samplewas the target protein. The protein identification was performed byDatabase searching using MASCOT (MatrixScience, London, UK) against NCBINR protein database.

N-Terminal Sequencing Via Edman Chemistry Degradation:

The N-terminal sequencing was performed on a Procise Protein Sequencer(model 494) from Applied Biosystems (Foster City, Calif.). The proteinsamples were separated first by SDS-PAGE, then blotted onto PVDFmembrane. The protein bands were excised from the membrane and loadedinto the Procise Sequencer. Eight cycles of Edman chemistry degradationwere run for each sample to get five AA residues at N-terminus. Astandard mix of 20 PTH-amino acids (Applied Biosystems) was run witheach sample. The amino acid residues from each Edman degradation weredetermined based on their retention times from the C-18 column againstthe standards.

The results of the MALDI sequencing indicated that the DGT-28 and DGT14proteins were expressed and that the TraP chimeric chloroplast transitpeptide sequences were processed. Table 34 lists the processed sequenceswhich were obtained by using the N-terminal Edman degradation and MALDIsequencing.

TABLE 34 Cleavage sites of TraP8 and TraP9 fused with thedgt-14 or dgt-28 coding sequences. The grey boxindicates the splice site. Number of Sequences with Construct SequenceSplicing TraP8-DG T14v2

66/67 TraP8-DG T28v1

66/67 TraP9-DG T14v2

62/63 TraP9-DG T28v1

—

What is claimed is:
 1. A nucleic acid molecule comprising apolynucleotide that encodes a chimeric protein comprising a syntheticchloroplast transit peptide (CTP) and a linked 5-enolpyruvylshikimate3-phosphate synthase (EPSPS) polypeptide, wherein the amino acidsequence of the synthetic CTP selected from the group consisting of: (A)a first amino acid sequence between 25 and 41 amino acids in length,wherein the first amino acid sequence is at least 90% identical to acontiguous amino acid sequence of the same length in the first 41 aminoacids of SEQ ID NO:1, linked to a second amino acid sequence between 25and 41 amino acids in length, wherein the second amino acid sequence isat least 90% identical to a contiguous amino acid sequence of the samelength in the last 41 amino acids of SEQ ID NO:2, and (B) a first aminoacid sequence between 25 and 41 amino acids in length, wherein the firstamino acid sequence is at least 90% identical to a contiguous amino acidsequence of the same length in the first 41 amino acids of SEQ ID NO:2,linked to a second amino acid sequence between 25 and 41 amino acids inlength, wherein the second amino acid sequence is at least 90% identicalto a contiguous amino acid sequence of the same length in the last 41amino acids of SEQ ID NO:1; and wherein the EPSPS polypeptide comprisesthe amino acid sequences of [SEQ ID NOs:170-173 from P11743.1US].
 2. Thenucleic acid molecule of claim 1, wherein the EPSPS polypeptidecomprises an amino acid sequence that is at least 90% identical to[DGT-28, DGT-31, DGT-32, or DGT-33].
 3. The nucleic acid molecule ofclaim 2, wherein the EPSPS polypeptide comprises an amino acid sequencethat is at least 95% identical to [DGT-28, DGT-31, DGT-32, or DGT-33].4. The nucleic acid molecule of claim 2, wherein the chimeric proteincomprises the amino acid sequence encoded by SEQ ID NO:34 or SEQ IDNO:35.
 5. The nucleic acid molecule of claim 1, wherein the syntheticCTP comprises SEQ ID NO:13.
 6. The nucleic acid molecule of claim 1,wherein the synthetic CTP is less than 70 amino acids in length.
 7. Thenucleic acid molecule of claim 1, wherein the synthetic CTP is about 65amino acids in length.
 8. The nucleic acid molecule of claim 2, whereinthe synthetic CTP comprises less than 7 conservative amino acidsubstitutions to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4,such that the synthetic CTP is at least 90% identical to SEQ ID NO:3 orSEQ ID NO:4.
 9. The nucleic acid molecule of claim 2, wherein thesynthetic CTP comprises less than 4 conservative amino acidsubstitutions to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4,such that the synthetic CTP is at least 95% identical to SEQ ID NO:3 orSEQ ID NO:4.
 10. The nucleic acid molecule of claim 2, wherein thesynthetic CTP comprises 1 conservative amino acid substitution to theamino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, such that thesynthetic CTP is at least 98% identical to SEQ ID NO:3 or SEQ ID NO:4.11. The nucleic acid molecule of claim 2, wherein the first amino acidsequence of the CTP comprises amino acids 1-35 of SEQ ID NO:1 or aminoacids 1-31 of SEQ ID NO:2.
 12. The nucleic acid molecule of claim 2,wherein the second amino acid sequence of the CTP comprises amino acids32-61 of SEQ ID NO:2 or amino acids 36-69 of SEQ ID NO:1.
 13. Thenucleic acid molecule of claim 2, wherein the polynucleotide is operablylinked to a promoter operable in a plant cell.
 14. The nucleic acidmolecule of claim 8, wherein the polynucleotide is operably linked to apromoter operable in a plant cell.
 15. The nucleic acid molecule ofclaim 14, wherein the molecule is a plant expression vector.
 16. Atransgenic plant material comprising the nucleic acid molecule of claim14 as a genomic nucleic acid molecule, wherein the EPSPS polypeptide istargeted to chloroplasts of the plant material.
 17. The transgenic plantmaterial of claim 16, wherein the plant material is selected from thegroup consisting of a plant cell, a plant tissue, a plant tissueculture, a callus culture, and a plant part.
 18. The transgenic plantmaterial of claim 16, wherein the plant material is a whole plant orseed.
 19. The transgenic plant material of claim 18, wherein the plantmaterial is from a plant selected from the group consisting ofArabidopsis, alfalfa, Brassica, beans, broccoli, cabbage, carrot,cauliflower, celery, Chinese cabbage, cotton, cucumber, eggplant,lettuce, melon, pea, pepper, peanut, potato, pumpkin, radish, rapeseed,spinach, soybean, squash, sugarbeet, sunflower, tobacco, tomato,watermelon, corn, onion, rice, sorghum, wheat, rye, millet, sugarcane,oat, triticale, switchgrass, and turfgrass.
 20. A method for producing atransgenic plant material, the method comprising transforming a plantmaterial with the nucleic acid molecule of claim
 15. 21. The methodaccording to claim 20, wherein the plant material is selected from thegroup consisting of a plant cell, a plant tissue, a plant tissueculture, and a callus culture.
 22. A transgenic plant commodity productproduced from the plant material of claim 16, wherein the commodityproduct comprises the polynucleotide.
 23. A method for growing aglyphosate-tolerant plant, the method comprising: planting orcultivating the transgenic plant or seed of claim 18 in a suitablegrowing environment.
 24. The method according to claim 23, the methodfurther comprising: applying glyphosate to the growing environment.